Thrombopoietic compounds

ABSTRACT

The invention relates to the field of compounds, especially peptides or polypeptides, that have thrombopoietic activity. The peptides and polypeptides of the invention may be used to increase platelets or platelet precursors (e.g., megakaryocytes) in a mammal.

This application is a divisional application of U.S. patent applicationSer. No. 13/278,137, which was filed Oct. 20, 2011, which is adivisional of U.S. patent application Ser. No. 10/933,133, now issuedU.S. Pat. No. 8,044,174, which was filed Sep. 2, 2004, which is acontinuation of U.S. patent application Ser. No. 09/422,838, now issuedU.S. Pat. No. 6,835,809, which was filed Oct. 22, 1999, which in turnclaims benefit under 35 U.S.C. §119 of U.S. Provisional PatentApplication Ser. No. 60/105,348, which was filed Oct. 23, 1998, each ofwhich is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledA-533-US-DIV4_SeqList_ST25.txt, created Apr. 29, 2014, which is 42 KB insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Generally, the invention relates to the field of compounds, especiallypeptides and polypeptides, that have thrombopoietic activity. Thecompounds of the invention may be used to increase production ofplatelets or platelet precursors (e.g., megakaryocytes) in a mammal

BACKGROUND OF THE INVENTION

This invention relates to compounds, especially peptides, that have theability to stimulate in vitro and in vivo production of platelets andtheir precursor cells such as megakaryocytes. Before discussing thenature of the inventive compounds, the following is provided as abackground regarding two proteins that have thrombopoietic activity:thrombopoietin (TPO) and megakaryocyte growth and development factor(MGDF).

The cloning of endogenous thrombopoietin (TPO) (Lok et al., Nature369:568-571 (1994); Bartley et al., Cell 77:1117-1124 (1994); Kuter etal., Proc. Natl. Acad. Sci. USA 91:11104-11108 (1994); de Sauvage etal., Nature 369:533- 538 (1994); Kato et al., Journal of Biochemistry119:229-236 (1995); Chang et al., Journal of Biological Chemistry270:511-514 (1995)) has rapidly increased our understanding ofmegakaryopoiesis (megakaryocyte production) and thrombopoiesis (plateletproduction).

Endogenous human TPO, a 60 to 70 kDa glycosylated protein primarilyproduced in the liver and kidney, consists of 332 amino acids (Bartleyet al., Cell 77:1117-1124 (1994); Chang et al., Journal of BiologicalChemistry 270:511-514 (1995)). The protein is highly conserved betweendifferent species, and has 23% homology with human erythropoietin(Gurney et al., Blood 85:981-988 (1995)) in the amino terminus (aminoacids 1 to 172) (Bartley et al., Cell 77:1117-1124 (1994)). EndogenousTPO has been shown to possess all of the characteristics of the keybiological regulator of thrombopoiesis. Its in vitro actions includespecific induction of megakaryocyte colonies from both purified murinehematopoietic stem cells (Zeigler et al., Blood 84:4045-4052 (1994)) andhuman CD34⁺ cells (Lok et al., Nature 369:568-571 (1994); Rasko et al.,Stem Cells 15:33-42 (1997)), the generation of megakaryocytes withincreased ploidy (Broudy et al., Blood 85:402-413 (1995)), and theinduction of terminal megakaryocyte maturation and platelet production(Zeigler et al., Blood 84:4045-4052 (1994); Choi et al., Blood85:402-413 (1995)). Conversely, synthetic antisenseoligodeoxynucleotides to the

TPO receptor (c-Mpl) significantly inhibit the colony-forming ability ofmegakaryocyte progenitors (Methia et al., Blood 82:1395-1401 (1993)).Moreover, c-Mpl knock-out mice are severely thrombocytopenic anddeficient in megakaryocytes (Alexander et al., Blood 87:2162-2170(1996)).

Recombinant human MGDF (rHuMGDF, Amgen Inc., Thousand Oaks, CA) isanother thrombopoietic polypeptide related to TPO. It is produced usingE. coli transformed with a plasmid containing cDNA encoding a truncatedprotein encompassing the amino-terminal receptor-binding domain of humanTPO (Ulich et al., Blood 86:971-976 (1995)). The polypeptide isextracted, refolded, and purified, and a poly[ethylene glycol] (PEG)moiety is covalently attached to the amino terminus The resultingmolecule is referred to herein as PEG-rHuMGDF or MGDF for short.

Various studies using animal models (Ulich, T.R. et al., Blood86:971-976 (1995); Hokom, M. M. et al., Blood 86:4486-4492 (1995)) haveclearly demonstrated the therapeutic efficacies of TPO and MGDF in bonemarrow transplantation and in the treatment of thrombocytopenia, acondition that often results from chemotherapy or radiation therapy.Preliminary data in humans have confirmed the utility of MGDF inelevating platelet counts in various settings. (Basser et al., Lancet348:1279-81 (1996); Kato et al., Journal of Biochemistry 119:229-236(1995); Ulich et al., Blood 86:971-976 (1995)). MGDF might be used toenhance the platelet donation process, since administration of MGDFincreases circulating platelet counts to about three-fold the originalvalue in healthy platelet donors.

TPO and MGDF exert their action through binding to the c-Mpl receptorwhich is expressed primarily on the surface of certain hematopoieticcells, such as megakaryocytes, platelets, CD34⁺ cells and primitiveprogenitor cells (Debili, N. et al., Blood 85:391-401 (1995); deSauvage, F. J. et al, Nature 369:533-538 (1994); Bartley, T. D., et al.,Cell 77:1117-1124 (1994); Lok, S. et al., Nature 369: 565-8 (1994)).Like most receptors for interleukins and protein hormones, c-Mpl belongsto the class I cytokine receptor superfamily (Vigon, I. et al., Proc.Natl. Acad. Sci. USA 89:5640-5644 (1992)). Activation of this class ofreceptors involves ligand-binding induced receptor homodimerizationwhich in turn triggers the cascade of signal transducing events.

In general, the interaction of a protein ligand with its receptor oftentakes place at a relatively large interface. However, as demonstrated inthe case of human growth hormone bound to its receptor, only a few keyresidues at the interface actually contribute to most of the bindingenergy (Clackson, T. et al., Science 267:383-386 (1995)). This and thefact that the bulk of the remaining protein ligand serves only todisplay the binding epitopes in the right topology makes it possible tofind active ligands of much smaller size.

In an effort toward this, the phage peptide library display system hasemerged as a powerful technique in identifying small peptide mimetics oflarge protein ligands (Scott, J. K. et al., Science 249:386 (1990);Devlin, J. J. et al., Science 249:404 (1990)). By using this technique,small peptide molecules that act as agonists of the c-Mpl receptor werediscovered (Cwirla, S. E. et al., Science 276:1696-1699 (1997)). In sucha study, random small peptide sequences displayed as fusions to the coatproteins of filamentous phage were affinity-eluted against the antibody-immobilized extracellular domain of c-Mpl and the retained phages wereenriched for a second round of affinity purification. This bindingselection and repropagation process was repeated many times to enrichthe pool of tighter binders. As a result, two families of c-Mpl-bindingpeptides, unrelated to each other in their sequences, were firstidentified. Mutagenesis libraries were then created to further optimizethe best binders, which finally led to the isolation of a very activepeptide with an IC₅₀=2 nM and an EC₅₀=400 nM (Cwirla, S. E. et al.,Science 276:1696-1699 (1997)). This 14-residue peptide, designated as aTMP (for TPO Mimetic Peptide), has no apparent sequence homology to TPOor MGDF. The structure of this TMP compound is as follows:

SEQ ID NO: 1 Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala orSEQ ID NO: 1 IEGPTLRQWLAARA using single letter amino acid abbreviations.

Previously, in a similar study on EPO mimetic peptides, an EPO mimeticpeptide (EMP) was discovered using the same technique (Wrighton, N. C.et al.,

Science 273:458-463 (1996)), and was found to act as a dimer in bindingto the EPO receptor (EPOR). The (ligand)₂/(receptor)₂ complex thusformed had a C2 symmetry according to X-ray crystallographic data(Livnah, O. et al., Science 273:464-471 (1996)). Based on thisstructural information, a covalently linked dimer of EMP in which theC-termini of two EMP monomers were crosslinked with a flexible spacerwas designed and found to have greatly enhanced binding as well as invitro/in vivo bioactivity (Wrighton, N. C., et al., Nature Biotechnology15:1261-1265 (1997)).

A similar C-terminal dimerization strategy was applied to the TPOmimetic peptide (TMP) (Cwirla, S. E. et al., Science 276:1696-1699(1997)). It was found that a C-terminally linked dimer (C-C link) of TMPhad an improved binding affinity of 0.5 nM and a remarkably increased invitro activity (EC₅₀=0.1 nM) in cell proliferation assays (Cwirla, S. E.et al., Science 276:1696-1699 (1997)). The structure of this TMP C-Cdimer is shown below: (SEQ ID N0:2)

In another aspect of the present invention, the tandem dimers may befurther attached to one or more moieties that are derived fromimmunoglobulin proteins, referred to generally as the Fc region of suchimmunoglobulins. The resulting compounds are referred to as Fc fusionsof TMP tandem dimers.

The following is a brief background section relating to the Fc regionsof antibodies that are useful in connection with some of the presentcompounds.

Antibodies comprise two functionally independent parts, a variabledomain known as “Fab”, which binds antigen, and a constant domain, knownas “Fc” which provides the link to effector functions such as complementfixation or phagocytosis. The Fc portion of an immunoglobulin has a longplasma half-life, whereas the Fab is short-lived. (Capon, et al., Nature337:525-531 (1989)).

Therapeutic protein products have been constructed using the Fc domainto attempt to provide longer half-life or to incorporate functions suchas Fc receptor binding, protein A binding, complement fixation, andplacental transfer which all reside in the Fc region of immunoglobulins(Capon, et al., Nature 337:525-531 (1989)). For example, the Fc regionof an IgG1 antibody has been fused to CD30-L, a molecule which bindsCD30 receptors expressed on Hodgkin's Disease tumor cells, anaplasticlymphoma cells, T-cell leukemia cells and other malignant cell types.See, U.S. Pat. No. 5,480,981. IL-10, an anti-inflammatory andantirejection agent has been fused to murine Fcγ2a in order to increasethe cytokine's short circulating half-life (Zheng, X. et al., TheJournal of Immunology, 154: 5590-5600 (1995)). Studies have alsoevaluated the use of tumor necrosis factor receptor linked with the Fcprotein of human IgG1 to treat patients with septic shock (Fisher, C. etal., N. Engl. J. Med., 334: 1697-1702 (1996); Van Zee, K. et al., TheJournal of Immunology, 156: 2221-2230 (1996)). Fc has also been fusedwith CD4 receptor to produce a therapeutic protein for treatment ofAIDS. See, Capon et al., Nature, 337:525-531 (1989). In addition,interleukin 2 has been fused to the Fc portion of IgG1 or IgG3 toovercome the short half life of interleukin 2 and its systemic toxicity.See, Harvill et al., Immunotechnology, 1: 95-105 (1995).

In spite of the availability of TPO and MGDF, there remains a need toprovide additional compounds that have a biological activity ofstimulating the production of platelets (thrombopoietic activity) and/orplatelet precursor cells, especially megakaryocytes (megakaryopoieticactivity). The present invention provides new compounds having suchactivity(ies), and related aspects.

SUMMARY OF THE INVENTION

The present invention provides a group of compounds that are capable ofbinding to and triggering a transmembrane signal through, i.e.,activating, the c-Mpl receptor, which is the same receptor that mediatesthe activity of endogenous thrombopoietin (TPO). Thus, the inventivecompounds have thrombopoietic activity,

i.e., the ability to stimulate, in vivo and in vitro, the production ofplatelets, and/or megakaryocytopoietic activity, i.e., the ability tostimulate, in vivo and in vitro, the production of platelet precursors.

In a first preferred embodiment, the inventive compounds comprise thefollowing general structure:

TMP₁-(L₁)_(n)-TMP₂

wherein TMP₁ and TMP₂ are each independently selected from the group ofcompounds comprising the core structure:

X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀,

wherein,

X₂ is selected from the group consisting of Glu, Asp, Lys, and Val;

X₃ is selected from the group consisting of Gly and Ala;

X₄ is Pro;

X₅ is selected from the group consisting of Thr and Ser;

X₆ is selected from the group consisting of Leu, Ile, Val, Ala, and Phe;

X₇ is selected from the group consisting of Arg and Lys;

X₈ is selected from the group consisting of Gln, Asn, and Glu;

X₉ is selected from the group consisting of Trp, Tyr, Cys, Ala, and Phe;

X₁₀ is selected from the group consisting of Leu, Ile, Val, Ala, Phe,Met, and Lys;

L₁ is a linker as described herein; and

n is 0 or 1;

and physiologically acceptable salts thereof.

In one embodiment, L₁ comprises (Gly)_(n), wherein n is 1 through 20,and when n is greater than 1, up to half of the Gly residues may besubstituted by another amino acid selected from the remaining 19 naturalamino acids or a stereoisomer thereof.

In addition to the core structure X₂-X₁₀ set forth above for TMP₁ andTMP₂, other related structures are also possible wherein one or more ofthe following is added to the TMP₁ and/or TMP₂ core structure: X₁ isattached to the N-terminus and/or X₁₁, X₁₂, X₁₃, and/or X₁₄ are attachedto the C-terminus, wherein X₁, X₁₂, X₁₃, and X₁₄ are as follows:

X₁ is selected from the group consisting of Ile, Ala, Val, Leu, Ser, andArg;

X₁₁ is selected from the group consisting of Ala, Ile, Val, Leu, Phe,Ser, Thr, Lys, His, and Glu;

X₁₂ is selected from the group consisting of Ala, Ile, Val, Leu, Phe,Gly, Ser, and Gln;

X₁₃ is selected from the group consisting of Arg, Lys, Thr, Val, Asn,Gln, and Gly; and

X₁₄ is selected from the group consisting of Ala, Ile, Val, Leu, Phe,Thr, Arg, Glu, and Gly.

In a second preferred embodiment, the inventive compounds have thegeneral formula:

(Fc)_(m)-(L₂)_(q)-TMP₁-(L₁)_(n)-TMP₂-(L₃)_(r)-(Fc)_(p)

wherein TMP₁, TMP₂ and n are each as described above; L₁, L₂ and L₃ arelinker groups which are each independently selected from the linkergroups described herein;

-   Fc is an Fc region of an immunoglobulin (as defined herein below);    m, p, q and r are each independently selected from the group    consisting of 0 and 1, wherein at least one of m or p is 1, and    further wherein if m is 0 then q is 0, and if p is 0, then r is 0;    and physiologically acceptable salts thereof In one embodiment, L₁,    L₂, and L₃ independently comprise (Gly)_(n), wherein n is 1 through    20, and when n is greater than 1, up to half of the Gly residues may    be substituted by another amino acid selected from the remaining 19    natural amino acids or a stereoisomer thereof.

Derivatives of the above compounds (described below) are alsoencompassed by this invention.

The compounds of this invention are preferably peptides, and they may beprepared by standard synthetic methods or any other methods of preparingpeptides. The compounds of this invention that encompass non-peptideportions may be synthesized by standard organic chemistry reactions, inaddition to standard peptide chemistry reactions when applicable.

The compounds of this invention may be used for therapeutic orprophylactic purposes by incorporating them with appropriatepharmaceutical carrier materials and administering an effective amountto a subject, such as a human (or other mammal). Other related aspectsare also included in the instant invention.

BRIEF DESCRIPTION OF THE FIGURES

Numerous other aspects and advantages of the present invention willtherefore be apparent upon consideration of the following detaileddescription thereof, reference being made to the drawings wherein:

FIGS. 1A and 1B show exemplary Fc polynucleotide and protein sequences(SEQ ID NO: 3 is the coding strand reading 5′→3′, SEQ ID NO: 4 is thecomplementary strand reading 3′→5′; and SEQ ID NO: 5 is the encodedamino acids sequence) of human IgG1 that may be used in the Fc fusioncompounds of this invention.

FIG. 2 shows a synthetic scheme for the preparation of pegylated peptide19 (SEQ ID NO:17).

FIG. 3 shows a synthetic scheme for the preparation of pegylated peptide20 (SEQ ID NO:18).

FIG. 4 shows the number of platelets generated in vivo in normal femaleBDF1 mice treated with one 100 μg/kg bolus injection of variouscompounds, as follows: PEG-MGDF means 20 kD average molecular weight PEGattached to the N-terminal amino group by reductive amination of aminoacids 1-163 of native human TPO, which is expressed in E. coli (so thatit is not glycosylated) (See WO 95/26746 entitled “Compositions andMethods for Stimulating Megakaryocyte Growth and Differentiation”); TMPmeans the compound of SEQ ID NO: 1; TMP-TMP means the compound of SEQ IDNO: 21; PEG-TMP-TMP means the compound of SEQ ID NO: 18, wherein the PEGgroup is a 5 kD average molecular weight PEG attached as shown in FIG.3; TMP-TMP-Fc is defined herein below and Fc-TMP-TMP is the same asTMP-TMP-Fc except that the Fc group is attached at the N-terminal endrather than the C-terminal end of the TMP-TMP peptide.

FIG. 5 shows the number of platelets generated in vivo in normal BDF1mice treated with various compounds delivered via implanted osmoticpumps over a 7-day period. The compounds are defined in the same manneras set forth above for FIG. 4.

FIGS. 6A, 6B, and 6C show schematic diagrams of preferred compounds ofthe present invention. FIG. 6A shows an Fc fusion compound wherein theFc moiety is fused at the N-terminus of the TMP dimer, and wherein theFc portion is a monomeric (single chain) form. FIG. 6B shows an Fcfusion compound wherein the Fc region is fused at the N-terminus of theTMP dimer, and wherein the Fc portion is dimeric, and one Fc monomer isattached to a TMP dimer. FIG. 6C shows an Fc fusion compound wherein theFc moiety is fused at the N-terminus of the TMP dimer, and wherein theFc portion is dimeric and each Fc monomer is attached to a TMP dimer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In an effort to seek small structures as lead compounds in thedevelopment of therapeutic agents with more desirable properties, adifferent type of dimer of TMP and related structures were designed inwhich the C-terminus of one TMP peptide was linked to the N-terminus ofa second TMP peptide, either directly or via a linker and the effects ofthis dimerization strategy on the bioactivity of the resulting dimericmolecules were then investigated. In some cases, these so-called tandemdimers (C—N link) were designed to have linkers between the twomonomers, the linkers being preferably composed of natural amino acids,therefore rendering their synthesis accessible to recombinanttechnologies.

The present invention is based on the discovery of a group of compoundsthat have thrombopoietic activity and which are thought to exert theiractivity by binding to the endogenous TPO receptor, c-Mpl.

Compounds and Derivatives

In a first preferred embodiment, the inventive compounds comprise thefollowing general structure:

TMP₁-(L₁)_(n)-TMP₂

wherein TMP₁ and TMP₂ are each independently selected from the group ofcompounds comprising the core structure:

X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀,

wherein,

X₂ is selected from the group consisting of Glu, Asp, Lys, and Val;

X₃ is selected from the group consisting of Gly and Ala;

X₄ is Pro;

X₅ is selected from the group consisting of Thr and Ser;

X₆ is selected from the group consisting of Leu, Ile, Val, Ala, and Phe;

X₇ is selected from the group consisting of Arg and Lys;

X₈ is selected from the group consisting of Gln, Asn, and Glu;

X₉ is selected from the group consisting of Trp, Tyr, and Phe;

X₁₀ is selected from the group consisting of Leu, Ile, Val, Ala, Phe,Met, and Lys;

L₁ is a linker as described herein; and

n is 0 or 1;

and physiologically acceptable salts thereof.

In one embodiment, L₁, comprises (Gly)_(n), wherein n is 1 through 20,and when n is greater than 1, up to half of the Gly residues may besubstituted by another amino acid selected from the remaining 19 naturalamino acids or a stereoisomer thereof.

In addition to the core structure X₂-X₁₀ set forth above for TMP₁ andTMP₂ , other related structures are also possible wherein one or more ofthe following is added to the TMP₁ and/or TMP₂ core structure: X₁ isattached to the N-terminus and/or X₁₁, X₁₂, X₁₃, and/or X₁₄ are attachedto the C-terminus, wherein X₁, X₁₁, X₁₂, X₁₃, and X₁₄ are as follows:

X₁ is selected from the group consisting of Ile, Ala, Val, Leu, Ser, andArg;

X₁₁ is selected from the group consisting of Ala, Ile, Val, Leu, Phe,Ser,

Thr, Lys, His, and Glu;

X₁₂ is selected from the group consisting of Ala, Ile, Val, Leu, Phe,Gly, Ser, and Gln;

X₁₃ is selected from the group consisting of Arg, Lys, Thr, Val, Asn,Gln, and Gly; and

X₁₄ is selected from the group consisting of Ala, Ile, Val, Leu, Phe,Thr, Arg, Glu, and Gly.

The term “TMP” is used to mean a moiety made up of, i.e., comprising, atleast 9 subunits (X₂-X₁₀), wherein X₂-X₁₀ comprise the core structure.The X₂-X₁₄ subunits are preferably amino acids independently selectedfrom among the 20 naturally-occurring amino acids, however, theinvention embraces compounds where X₂-X₁₄ are independently selectedfrom the group of atypical, non-naturally occurring amino acids wellknown in the art. Specific preferred amino acids are identified for eachposition. For example, X₂ may be Glu, Asp, Lys, or Val. Boththree-letter and single letter abbreviations for amino acids are usedherein; in each case, the abbreviations are the standard ones used forthe 20 naturally-occurring amino acids or well-known variations thereofThese amino acids may have either L or D stereochemistry (except forGly, which is neither L nor D), and the TMPs may comprise a combinationof stereochemistries. However, the L stereochemistry is preferred forall of the amino acids in the TMP chain. The invention also providesreverse TMP molecules wherein the amino terminal to carboxy terminalsequence of the amino acids is reversed. For example, the reverse of amolecule having the normal sequence X₁-X₂-X₃ would be X₃-X₂-X₁. Theinvention also provides retro-reverse TMP molecules wherein, like areverse TMP, the amino terminal to carboxy terminal sequence of aminoacids is reversed and residues that are normally “L” enatiomers in TMPare altered to the “D” stereoisomer form.

Additionally, physiologically acceptable salts of the TMPs are alsoencompassed. “Physiologically acceptable salts” means any salts that areknown or later discovered to be pharmaceutically acceptable. Somespecific preferred examples are: acetate, trifluoroacetate,hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate,oxalate.

It is also contemplated that “derivatives” of the TMPs may besubstituted for the above-described TMPs. Such derivative TMPs includemoieties wherein one or more of the following modifications have beenmade:

one or more of the peptidyl [—C(O)NR—] linkages (bonds) have beenreplaced by a non-peptidyl linkage such as a —CH₂-carbamate linkage[—CH₂—OC(O)NR—]; a phosphonate linkage; a —CH₂-sulfonamide[—CH₂—S(O)₂NR—] linkage; a urea [—NHC(O)NH—] linkage; a —CH₂-secondaryamine linkage; or an alkylated peptidyl linkage [—C(O)NR⁶— where R⁶ islower alkyl];

peptides wherein the N-terminus is derivatized to a —NRR¹ group; to a—NRC(O)R group; to a —NRC(O)OR group; to a —NRS(O)₂R group; to a—NHC(O)NHR group, where R and R¹ are hydrogen or lower alkyl, with theproviso that R and R¹ are not both hydrogen; to a succinimide group; toa benzyloxycarbonyl-NH— (CBZ-NH—) group; or to a benzyloxycarbonyl-NH—group having from 1 to 3 substituents on the phenyl ring selected fromthe group consisting of lower alkyl, lower alkoxy, chloro, and bromo;and

peptides wherein the free C terminus is derivatized to —C(O)R² where R²is selected from the group consisting of lower alkoxy and —NR³R⁴ whereR³ and R⁴ are independently selected from the group consisting ofhydrogen and lower alkyl. By “lower” is meant a group having from 1 to 6carbon atoms.

Additionally, modifications of individual amino acids may be introducedinto the TMP molecule by reacting targeted amino acid residues of thepeptide with an organic derivatizing agent that is capable of reactingwith selected side chains or terminal residues. The following areexemplary:

Lysinyl and amino terminal residues may be reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing alpha-amino-containing residuesinclude imidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues may be modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineguanidino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane may be used to form 0- acetyl tyrosyl species and3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modifiedby reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues may be deamidated under mildly acidic conditions. Either formof these residues falls within the scope of this invention.

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix or to other macromolecular carriers. Commonly used cross-linkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis (succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 may be employed for protein immobilization.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains(Creighton, T. E., Proteins: Structure and Molecule Properties, W. H.Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of theN-terminal amine, and, in some instances, amidation of the C-terminalcarboxyl groups.

Such derivatized moieties preferably improve one or more characteristicsincluding thrombopoietic activity, solubility, absorption, biologicalhalf life, and the like of the inventive compounds. Alternatively,derivatized moieties result in compounds that have the same, oressentially the same, characteristics and/or properties of the compoundthat is not derivatized. The moieties may alternatively eliminate orattenuate any undesirable side effect of the compounds and the like.

In addition to the core structure set forth above, X₂-X₁₀, otherstructures that are specifically contemplated are those in which one ormore additional X groups are attached to the core structure. Thus, X₁,and/or X₁₁, X₁₂, X₁₃, and X₁₄ may be attached to the core structure.Some exemplary additional structures are the following:

-   X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁;-   X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂;-   X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃;-   X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄;-   X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀;-   X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁;-   X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂;-   X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃;-   X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄,    wherein X₁ through X₁₄ are as described above. Each of TMP₁ and TMP₂    may be the same or different in sequence and/or length. In some    preferred embodiments, TMP₁ and TMP₂ are the same.

A particularly preferred TMP is the following:

Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala (SEQ ID NO:1).

As used herein “comprising” means, inter alia, that a compound mayinclude additional amino acids on either or both of the—or C-termini ofthe given sequence. However, as long as a structure such as X₂ to X₁₀,X₁ to X₁₄, or one of the other exemplary structures is present, theremaining chemical structure is relatively less important. Of course,any structure outside of the core X₂ to X₁₀ structure, or the X₁ to X₁₄,structure, should not significantly interfere with thrombopoieticactivity of the compound. For example, an N-terminal Met residue isenvisioned as falling within this invention. Additionally, although manyof the preferred compounds of the invention are tandem dimers in thatthey possess two TMP moieties, other compounds of this invention aretandem multimers of the TMPs, i.e., compounds of the following exemplarystructures:

TMP₁-L-TMP₂-L-TMP₃;

TMP₁-L-TMP₂-L-TMP₃-L-TMP₄;

TMP₁-L-TMP₂-L-TMP₃-L-TMP₄-L-TMP₅;

wherein TMP₁, TMP₂, TMP₃, TMP₄, and TMP₅ can have the same or differentstructures, and wherein each TMP and L is defined as set forth herein,and the linkers are each optional. Preferably, the compounds of thisinvention will have from 2-5 TMP moieties, particularly preferably 2-3,and most preferably 2. The compounds of the first embodiment of thisinvention will preferably have less than about 60, more preferably lessthan about 40 amino acids in total (i.e., they will be peptides).

As noted above, the compounds of the first embodiment of this inventionare preferably TMP dimers which are either bonded directly or are linkedby a linker group. The monomeric TMP moieties are shown in theconventional orientation from N to C terminus reading from left toright. Accordingly, it can be seen that the inventive compounds are alloriented so that the C terminus of TMP₁ is attached either directly orthrough a linker to the N-terminus of TMP₂. This orientation is referredto as a tandem orientation, and the inventive compounds may be generallyreferred to as “tandem dimers”. These compounds are referred to asdimers even if TMP₁ and TMP₂ are structurally distinct. That is, bothhomodimers and heterodimers are envisioned.

The “linker” group (“L₁”) is optional. When it is present, it is notcritical what its chemical structure is, since it serves primarily as aspacer. The linker should be chosen so as not to interfere with thebiological activity of the final compound and also so thatimmunogenicity of the final compound is not significantly increased. Thelinker is preferably made up of amino acids linked together by peptidebonds. Thus, in preferred embodiments, the linker comprises Y_(n),wherein Y is a naturally occurring amino acid or a steroisomer thereofand “n” is any one of 1 through 20. The linker is therefore made up offrom 1 to 20 amino acids linked by peptide bonds, wherein the aminoacids are selected from the 20 naturally-occurring amino acids. In amore preferred embodiment, the 1 to 20 amino acids are selected fromGly, Ala, Pro, Asn, Gln, Cys, Lys. Even more preferably, the linker ismade up of a majority of amino acids that are sterically un-hindered,such as Gly, Gly-Gly [(Gly)₂], Gly-Gly-Gly [(Gly)₃] . . . (Gly)₂₀, Ala,Gly-Ala, Ala-Gly, Ala-Ala, etc. Other specific examples of linkers are:

-   -   (Gly)₃Lys(Gly)₄ (SEQ ID NO: 6);    -   (Gly)₃AsnGlySer(Gly)₂ (SEQ ID NO: 7)    -   (this structure provides a site for glycosylation, when it is        produced recombinantly in a mammalian cell system that is        capable of glycosylating such sites);    -   (Gly)₃Cys(Gly)₄ (SEQ ID NO: 8); and    -   GlyProAsnGly (SEQ ID NO: 9).    -   To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄        means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. Combinations of Gly and        Ala are also preferred.

Non-peptide linkers are also possible. For example, alkyl linkers suchas —HN—(CH₂)_(s)—CO—, wherein s=2-20 could be used. These alkyl linkersmay further be substituted by any non-sterically hindering group such aslower alkyl (e.g., C₁-C₆), lower acyl, halogen (e.g., Cl, Br), CN, NH₂,phenyl, etc.

Another type of non-peptide linker is a polyethylene glycol group, suchas:

—HN—CH₂—CH₂—(O—CH₂—CH₂)_(n)—O—CH₂—CO—

wherein n is such that the overall molecular weight of the linker rangesfrom approximately 101 to 5000, preferably 101 to 500.

In general, it has been discovered that a linker of a length of about0-14 sub-units (e g , amino acids) is preferred for the thrombopoieticcompounds of the first embodiment of this invention.

The peptide linkers may be altered to form derivatives in the samemanner as described above for the TMPs.

The compounds of this first group may further be linear or cyclic. By“cyclic” is meant that at least two separated, i.e., non-contiguous,portions of the molecule are linked to each other. For example, theamino and carboxy terminus of the ends of the molecule could becovalently linked to form a cyclic molecule. Alternatively, the moleculecould contain two or more Cys residues (e.g., in the linker), whichcould cyclize via disulfide bond formation. It is further contemplatedthat more than one tandem peptide dimer can link to form a dimer ofdimers. Thus, for example, a tandem dimer containing a Cys residue canform an intermolecular disulfide bond with a Cys of another such dimer.See, for example, the compound of SEQ ID NO: 20, below.

The compounds of the invention may also be covalently or noncovalentlyassociated with a carrier molecule, such as a linear polymer (e.g.,polyethylene glycol, polylysine, dextran, etc.), a branched-chainpolymer (see, for example, U.S. Pat. No. 4,289,872 to Denkenwalter etal., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, issued Jul.20, 1993; WO 93/21259 by Frechet et al., published 28 Oct. 1993); alipid; a cholesterol group (such as a steroid); or a carbohydrate oroligosaccharide. Other possible carriers include one or more watersoluble polymer attachments such as polyoxyethylene glycol, orpolypropylene glycol as described U.S. Pat. Nos. 4,640,835, 4,496,689,4,301,144, 4,670,417, 4,791,192 and 4,179,337. Still other usefulpolymers known in the art include monomethoxy-polyethylene glycol,dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co- polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol, as well as mixtures of thesepolymers.

A preferred such carrier is polyethylene glycol (PEG). The PEG group maybe of any convenient molecular weight and may be straight chain orbranched. The average molecular weight of the PEG will preferably rangefrom about 2 kDa to about 100 kDa, more preferably from about 5 kDa toabout 50 kDa, most preferably from about 5 kDa to about 10 kDa.

The PEG groups will generally be attached to the compounds of theinvention via acylation, reductive alkylation, Michael addition, thiolalkylation or other chemoselective conjugation/ligation methods througha reactive group on the PEG moiety (e.g., an aldehyde, amino, ester,thiol, a-haloacetyl, maleimido or hydrazino group) to a reactive groupon the target compound (e.g., an aldehyde, amino, ester, thiol,a-haloacetyl, maleimido or hydrazino group).

Carbohydrate (oligosaccharide) groups may conveniently be attached tosites that are known to be glycosylation sites in proteins. Generally,0-linked oligosaccharides are attached to serine (Ser) or threonine(Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids not countingproline. The structures of N-linked and 0-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, may confer acidic properties to the glycosylated compound. Suchsite(s) may be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites may further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

Some exemplary peptides of this invention are shown below. Single letteramino acid abbreviations are used, and the linker is shown separated bydashes for clarity. Additional abbreviations: BrAc means bromoacetyl(BrCH₂C(O)) and PEG is polyethylene glycol.

In each of the above compounds, an N-terminal Met (or M residue, usingthe one-letter code) is contemplated as well. Multimers (e.g., tandemand non-tandem, covalently bonded and non-covalently bonded) of theabove compounds are also contemplated.

In a second embodiment of this invention, the compounds described abovemay further be fused to one or more Fc groups, either directly orthrough linker groups. In general, the formula of this second group ofcompounds is:

(Fc)_(m)-(L₂)_(q)-TMP₁-(L₁)_(n)-TMP₂-(L₃)_(r)-Fc)_(p)

wherein TMP₁, TMP₂ and n are each as described above; L₁, L₂ and L₃ arelinker groups which are each independently selected from the linkergroups described above; Fc is an Fc region of an immunoglobulin; m, p, qand r are each independently selected from the group consisting of 0 and1, wherein at least one of m or p is 1, and further wherein if m is 0then q is 0, and if p is 0, then r is 0; and physiologically acceptablesalts thereof

Accordingly, the compounds of this second group have structures asdefined for the first group of compounds as described above, but thesecompounds are further fused to at least one Fc group either directly orthrough one or more linker groups. The Fc sequence of the abovecompounds may be selected from the human immunoglobulin IgG-1 heavychain, see Ellison, J. W. et al., Nucleic Acids Res. 10:4071-4079(1982), or any other Fc sequence known in the art (e.g. other IgGclasses including but not limited to IgG-2, IgG-3 and IgG-4, or otherimmunoglobulins).

It is well known that Fc regions of antibodies are made up of monomericpolypeptide segments that may be linked into dimeric or multimeric formsby disulfide bonds or by non-covalent association. The number ofintermolecular disulfide bonds between monomeric subunits of native Fcmolecules ranges from 1 to 4 depending on the class (e.g., IgG, IgA,IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2) of antibodyinvolved. The term “Fc” as used herein is generic to the monomeric,dimeric, and multimeric forms of Fc molecules. It should be noted thatFc monomers will spontaneously dimerize when the appropriate Cysresidues are present unless particular conditions are present thatprevent dimerization through disulfide bond formation. Even if the Cysresidues that normally form disulfide bonds in the Fc dimer are removedor replaced by other residues, the monomeric chains will generallydimerize through non- covalent interactions. The term “Fc” herein isused to mean any of these forms: the native monomer, the native dimer(disulfide bond linked), modified dimers (disulfide and/ornon-covalently linked), and modified monomers (i.e., derivatives).

Variants, analogs or derivatives of the Fc portion may be constructedby, for example, making various substitutions of residues or sequences.

Variant (or analog) polypeptides include insertion variants, wherein oneor more amino acid residues supplement an Fc amino acid sequence.Insertions may be located at either or both termini of the protein, ormay be positioned within internal regions of the Fc amino acid sequence.Insertional variants with additional residues at either or both terminican include for example, fusion proteins and proteins including aminoacid tags or labels. For example, the Fc molecule may optionally containan N-terminal Met, especially when the molecule is expressedrecombinantly in a bacterial cell such as E. coli.

In Fc deletion variants, one or more amino acid residues in an Fcpolypeptide are removed. Deletions can be effected at one or bothtermini of the Fc polypeptide, or with removal of one or more residueswithin the Fc amino acid sequence. Deletion variants, therefore, includeall fragments of an Fc polypeptide sequence.

In Fc substitution variants, one or more amino acid residues of an Fcpolypeptide are removed and replaced with alternative residues. In oneaspect, the substitutions are conservative in nature, however, theinvention embraces substitutions that ore also non-conservative.

For example, cysteine residues can be deleted or replaced with otheramino acids to prevent formation of some or all disulfide crosslinks ofthe Fc sequences. In particular, the amino acids at positions 7 and 10of SEQ ID NO:5 are cysteine residues. One may remove each of thesecysteine residues or substitute one or more such cysteine residues withother amino acids, such as Ala or Ser. As another example, modificationsmay also be made to introduce amino acid substitutions to (1) ablate theFc receptor binding site; (2) ablate the complement (Clq) binding site;and/or to (3) ablate the antibody dependent cell-mediated cytotoxicity(ADCC) site. Such sites are known in the art, and any knownsubstitutions are within the scope of Fc as used herein. For example,see Molecular Immunology, Vol. 29, No. 5, 633-639 (1992) with regards toADCC sites in IgG1.

Likewise, one or more tyrosine residues can be replaced by phenylalanineresidues as well. In addition, other variant amino acid insertions,deletions (e.g., from 1-25 amino acids) and/or substitutions are alsocontemplated and are within the scope of the present invention.Conservative amino acid substitutions will generally be preferred.Furthermore, alterations may be in the form of altered amino acids, suchas peptidomimetics or D-amino acids.

Fc sequences of the TMP compound may also be derivatized, i.e., bearingmodifications other than insertion, deletion, or substitution of aminoacid residues. Preferably, the modifications are covalent in nature, andinclude for example, chemical bonding with polymers, lipids, otherorganic and inorganic moieties. Derivatives of the invention may beprepared to increase circulating half-life, or may be designed toimprove targeting capacity for the polypeptide to desired cells,tissues, or organs.

It is also possible to use the salvage receptor binding domain of theintact Fc molecule as the Fc part of the inventive compounds, such asdescribed in WO 96/32478, entitled “Altered Polypeptides with IncreasedHalf-Life”. Additional members of the class of molecules designated asFc herein are those that are described in WO 97/34631, entitled“Immunoglobulin-Like Domains with Increased Half-Lives”. Both of thepublished PCT applications cited in this paragraph are herebyincorporated by reference.

The Fc fusions may be at the N or C terminus of TMP₁ or TMP₂ or at boththe N and C termini of the TMPs. It has been surprisingly discoveredthat peptides in which an Fc moiety is ligated to the N terminus of theTMP group is more bioactive than the other possibilities, so the fusionhaving an Fc domain at the N terminus of TMP₁ (i.e., r and p are both 0and m and q are both 1 in general formula) is preferred. When the Fcchain is fused at the N-terminus of the TMP or linker, such fusion willgenerally occur at the C-terminus of the Fc chain, and vice versa.

Also preferred are compounds that are dimers (e.g., tandem andnon-tandem) of the compounds set forth in the general formula as set outabove. In such cases, each Fc chain will be linked to a tandem dimer ofTMP peptides. A schematic example of such a compound is shown in FIG.6C. A preferred example of this type of compound is based on FIG. 6C,wherein Fc is a dimer of the compound of SEQ ID NO: 5, each L₂ is(Gly)₅, TMP₁ and TMP₂ are each the compound of SEQ ID NO: 1, and each L₁is (Gly)₈ . This compound is also referred to herein as“Fc-TMP₁-L-TMP₂”. It is also represented as a dimer (through the Fcportion) of SEQ ID NO: 34.

The analogous compound wherein the Fc group is attached through a linkerto the C-terminus of the TMP₂ groups in FIG. 6 C is also contemplatedand is referred to herein as TMP₁-L-TMP₂-Fc.

Some specific examples of compounds from the second group are providedas follows:

In each of the above compounds, an additional N-terminal Met (or Mresidue, using the one-letter code) is contemplated as well. The Metresidue may be attached at the N-terminus of the Fc group in those caseswherein there is an Fc group attached to the N-terminus of the TMP. Inthose cases wherein the Fc group is attached at the C-terminus of theTMP, the Met residues could be attached to the N-terminus of the TMPgroup.

In each of the above cases Fc is preferably the Fc region of the humanimmunoglobulin IgG1 heavy chain or a biologically active fragment,derivative, or dimer thereof, see Ellison, J. W. et al., Nucleic AcidsRes. 10:4071-4079 (1982).

The Fc sequence shown in SEQ ID NO: 5 is the most preferred Fc for theabove compounds. Also preferred are the above compounds in which the Fcis a dimeric form of the sequence of SEQ ID NO: 5 and each Fc chain isattached to a TMP tandem dimer.

Additionally, although many of the preferred compounds of the secondembodiment include one or more tandem dimers in that they possess twolinked TMP moieties, other compounds of this invention include tandemmultimers of the TMPs, i.e., compounds of the following exemplarystructures:

Fc-TMP₁-L-TMP₂-L-TMP₃;

Fc-TMP₁-L-TMP₂-L-TMP₃-L-TMP₄;

Fc-TMP₁-L-TMP₂-L-TMP₃-L-TMP₄-L-TMP₅;

TMP₁-L-TMP₂-L-TMP₃-L-Fc;

TMP₁-L-TMP₂-L-TMP₃-L-TMP₄-L-Fc;

TMP₁-L-TMP₂-L-TMP₃-L-TMP₄-L-TMP_(—5)-L-Fc;

wherein TMP₁, TMP₂, TMP₃, TMP₄, and TMP₅ can have the same or differentstructures, and wherein Fc and each TMP and L is defined as set forthabove, and the linkers are each optional. In each case above, the Fcgroup can be monomeric or dimeric, and in cases where the Fc is dimeric,one or more TMP multimer can be attached to each Fc chains. Alsocontemplated are other examples wherein the TMP dimers or multimers areattached to both the N and C-termini of one or both Fc chains, includingthe case wherein TMP dimers or multimers are attached to all fourtermini of two Fc chains.

Preferably, the compounds of this second embodiment of the inventionwill have from about 200 to 400 amino acids in total (i.e., they will bepolypeptides).

Methods of Making

The compounds of this invention may be made in a variety of ways. Sincemany of the compounds will be peptides, or will include a peptide,methods for synthesizing peptides are of particular relevance here. Forexample, solid phase synthesis techniques may be used. Suitabletechniques are well known in the art, and include those described inMerrifield, in Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotiseds. 1973); Merrifield, J. Am. Chem. Soc. 85:2149 (1963); Davis et al.,Biochem. Intl. 10:394-414 (1985); Stewart and Young, Solid Phase PeptideSynthesis (1969); U.S. Pat. No. 3,941,763; Finn et al., The Proteins,3rd ed., vol. 2, pp. 105-253 (1976); and Erickson et al., The Proteins,3rd ed., vol. 2, pp. 257-527 (1976). Solid phase synthesis is thepreferred technique of making individual peptides since it is the mostcost-effective method of making small peptides.

The peptides may also be made in transformed host cells usingrecombinant DNA techniques. To do so, a recombinant DNA molecule codingfor the peptide is prepared. Methods of preparing such DNA and/or RNAmolecules are well known in the art. For instance, sequences coding forthe peptides could be excised from DNA using suitable restrictionenzymes. The relevant sequences can be created using the polymerasechain reaction (PCR) with the inclusion of useful restriction sites forsubsequent cloning. Alternatively, the DNA/RNA molecule could besynthesized using chemical synthesis techniques, such as thephosphoramidite method. Also, a combination of these techniques could beused.

The invention also includes a vector encoding the peptides in anappropriate host. The vector comprises the DNA molecule that encodes thepeptides operatively linked to appropriate expression control sequences.Methods of effecting this operative linking, either before or after thepeptide-encoding DNA molecule is inserted into the vector, are wellknown. Expression control sequences include promoters, activators,enhancers, operators, ribosomal binding sites, start signals, stopsignals, cap signals, polyadenylation signals, and other signalsinvolved with the control of transcription or translation.

The resulting vector comprising the peptide-encoding DNA molecule isused to transform an appropriate host. This transformation may beperformed using methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These factorsinclude, for example, compatibility with the chosen expression vector,toxicity to the host cell of the peptides encoded by the DNA molecule,rate of transformation, ease of recovery of the peptides, expressioncharacteristics, bio-safety and costs. A balance of these factors mustbe struck with the understanding that not all hosts may be equallyeffective for the expression of a particular DNA sequence.

Within these general guidelines, useful microbial hosts include bacteria(such as E. coli), yeast (such as Saccharomyces sp. and Pichia pastoris)and other fungi, insects, plants, mammalian (including human) cells inculture, or other hosts known in the art.

Next, the transformed host is cultured under conventional fermentationconditions so that the desired peptides are expressed. Such fermentationconditions are well known in the art.

Finally, the peptides are purified from the fermentation culture or fromthe host cells in which they are expressed. These purification methodsare also well known in the art.

Compounds that contain derivatized peptides or which contain non-peptidegroups may be synthesized by well-known organic chemistry techniques.

Uses of the Compounds

The compounds of this invention have the ability to bind to and activatethe c-Mpl receptor, and/or have the ability to stimulate the production(both in vivo and in vitro) of platelets (“thrombopoietic activity”) andplatelet precursors (“megakaryocytopoietic activity”). To measure theactivity (-ies) of these compounds, one can utilize standard assays,such as those described in WO95/26746 entitled “Compositions and Methodsfor Stimulating Megakaryocyte Growth and Differentiation”. In vivoassays are further described in the Examples section herein.

The conditions to be treated by the methods and compositions of thepresent invention are generally those which involve an existingmegakaryocyte/platelet deficiency or an expected or anticipatedmegakaryocyte/platelet deficiency in the future (e.g., because ofplanned surgery or platelet donation). Such conditions may be the resultof a deficiency (temporary or permanent) of active Mpl ligand in vivo.The generic term for platelet deficiency is thrombocytopenia, and hencethe methods and compositions of the present invention are generallyavailable for prophylactically or therapeutically treatingthrombocytopenia in patients in need thereof.

The World Health Organization has classified the degree ofthrombocytopenia on the number of circulating platelets in theindividual (Miller, et al., Cancer 47:210-211 (1981)). For example, anindividual showing no signs of thrombocytopenia (Grade 0) will generallyhave at least 100,000 platelets/mm³ Mild thrombocytopenia (Grade 1)indicates a circulating level of platelets between 79,000 and99,000/mm³. Moderate thrombocytopenia (Grade 2) shows between 50,000 and74,000 platelets/mm³ and severe thrombocytopenia is characterized bybetween 25,000 and 49,000 platelets/mm³. Life-threatening ordebilitating thrombocytopenia is characterized by a circulatingconcentration of platelets of less than 25,000/mm³.

Thrombocytopenia (platelet deficiencies) may be present for variousreasons, including chemotherapy and other therapy with a variety ofdrugs, radiation therapy, surgery, accidental blood loss, and otherspecific disease conditions. Exemplary specific disease conditions thatinvolve thrombocytopenia and may be treated in accordance with thisinvention are: aplastic anemia; idiopathic or immune thrombocytopenia(ITP), including idiopathic thrombocytopenic purpura associated withbreast cancer; HIV associated ITP and HIV-related thromboticthrombocytopenic purpura; metastatic tumors which result inthrombocytopenia; systemic lupus erythematosus; including neonatal lupussyndrome splenomegaly; Fanconi's syndrome; vitamin B12 deficiency; folicacid deficiency; May-Hegglin anomaly; Wiskott-Aldrich syndrome; chronicliver disease; myelodysplastic syndrome associated withthrombocytopenia; paroxysmal nocturnal hemoglobinuria; acute profoundthrombocytopenia following C7E3 Fab (Abciximab) therapy; alloimmunethrombocytopenia, including maternal alloimmune thrombocytopenia;thrombocytopenia associated with antiphospholipid antibodies andthrombosis; autoimmune thrombocytopenia; drug-induced immunethrombocytopenia, including carboplatin-induced thrombocytopenia,heparin-induced thrombocytopenia; fetal thrombocytopenia; gestationalthrombocytopenia; Hughes' syndrome; lupoid thrombocytopenia; accidentaland/or massive blood loss; myeloproliferative disorders;thrombocytopenia in patients with malignancies; thromboticthrombocytopenia purpura, including thrombotic microangiopathymanifesting as thrombotic thrombocytopenic purpura/hemolytic uremicsyndrome in cancer patients; autoimmune hemolytic anemia; occult jejunaldiverticulum perforation; pure red cell aplasia; autoimmunethrombocytopenia; nephropathia epidemica; rifampicin-associated acuterenal failure; Paris-Trousseau thrombocytopenia; neonatal alloimmunethrombocytopenia; paroxysmal nocturnal hemoglobinuria; hematologicchanges in stomach cancer; hemolytic uremic syndromes in childhood;hematologic manifestations related to viral infection includinghepatitis A virus and CMV-associated thrombocytopenia. Also, certaintreatments for AIDS result in thrombocytopenia (e.g., AZT). Certainwound healing disorders might also benefit from an increase in plateletnumbers.

With regard to anticipated platelet deficiencies, e.g., due to futuresurgery, a compound of the present invention could be administeredseveral days to several hours prior to the need for platelets. Withregard to acute situations, e.g., accidental and massive blood loss, acompound of this invention could be administered along with blood orpurified platelets.

The compounds of this invention may also be useful in stimulatingcertain cell types other than megakaryocytes if such cells are found toexpress Mpl receptor. Conditions associated with such cells that expressthe Mpl receptor, which are responsive to stimulation by the Mpl ligand,are also within the scope of this invention.

The compounds of this invention may be used in any situation in whichproduction of platelets or platelet precursor cells is desired, or inwhich stimulation of the c-Mpl receptor is desired. Thus, for example,the compounds of this invention may be used to treat any condition in amammal wherein there is a need of platelets, megakaryocytes, and thelike. Such conditions are described in detail in the following exemplarysources: WO95/26746; WO95/21919; WO95/18858; WO95/21920 and areincorporated herein.

The compounds of this invention may also be useful in maintaining theviability or storage life of platelets and/or megakaryocytes and relatedcells. Accordingly, it could be useful to include an effective amount ofone or more such compounds in a composition containing such cells.

By “mammal” is meant any mammal, including humans, domestic animalsincluding dogs and cats; exotic and/or zoo animals including monkeys;laboratory animals including mice, rats, and guinea pigs; farm animalsincluding horses, cattle, sheep, goats, and pigs; and the like. Thepreferred mammal is human.

Pharmaceutical Compositions

The present invention also provides methods of using pharmaceuticalcompositions of the inventive compounds. Such pharmaceuticalcompositions may be for administration for injection, or for oral,nasal, transdermal or other forms of administration, including, e.g., byintravenous, intradermal, intramuscular, intramammary, intraperitoneal,intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., aerosolizeddrugs) or subcutaneous injection (including depot administration forlong term release); by sublingual, anal, vaginal, or by surgicalimplantation, e.g., embedded under the splenic capsule, brain, or in thecornea. The treatment may consist of a single dose or a plurality ofdoses over a period of time. In general, comprehended by the inventionare pharmaceutical compositions comprising effective amounts of acompound of the invention together with pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers. Such compositions include diluents of various buffer content(e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additivessuch as detergents and solubilizing agents (e.g., Tween 80, Polysorbate80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances(e.g., lactose, mannitol); incorporation of the material intoparticulate preparations of polymeric compounds such as polylactic acid,polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also beused, and this may have the effect of promoting sustained duration inthe circulation. The pharmaceutical compositions optionally may includestill other pharmaceutically acceptable liquid, semisolid, or soliddiluents that serve as pharmaceutical vehicles, excipients, or media,including but are not limited to, polyoxyethylene sorbitan monolaurate,magnesium stearate, methyl- and propylhydroxybenzoate, starches,sucrose, dextrose, gum acacia, calcium phosphate, mineral oil, cocoabutter, and oil of theobroma. Such compositions may influence thephysical state, stability, rate of in vivo release, and rate of in vivoclearance of the present proteins and derivatives. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated byreference. The compositions may be prepared in liquid form, or may be indried powder, such as lyophilized form. Implantable sustained releaseformulations are also contemplated, as are transdermal formulations.

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, K., Modern Pharmaceutics, Edited by G.S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated byreference. In general, the formulation will include the inventivecompound, and inert ingredients which allow for protection against thestomach environment, and release of the biologically active material inthe intestine.

Also specifically contemplated are oral dosage forms of the aboveinventive compounds. If necessary, the compounds may be chemicallymodified so that oral delivery is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe compound molecule itself, where said moiety permits (a) inhibitionof proteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecompound and increase in circulation time in the body. Examples of suchmoieties include: Polyethylene glycol, copolymers of ethylene glycol andpropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinyl pyrrolidone and polyproline (Abuchowski and Davis, SolublePolymer-Enzyme Adducts, Enzymes as Drugs, Hocenberg and Roberts, eds.,Wiley-Interscience, New York, N.Y., (1981), pp 367-383; Newmark, et al.,J. Appl. Biochem. 4:185-189 (1982)). Other polymers that could be usedare poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

For the oral delivery dosage forms, it is also possible to use a salt ofa modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhance absorption of thetherapeutic compounds of this invention. The clinical efficacy of aheparin formulation using SNAC has been demonstrated in a Phase II trialconducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, “Oraldrug delivery composition and methods”.

The therapeutic can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, theprotein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may also be used as fillersincluding calcium triphosphate, magnesium carbonate and sodium chloride.Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500,Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrants include but are notlimited to starch including the commercial disintegrant based on starch,Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment, asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethoniumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives which potentially enhance uptake of the compound are forinstance the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release formulation may be desirable. The drug could beincorporated into an inert matrix which permits release by eitherdiffusion or leaching mechanisms e.g., gums. Slowly degeneratingmatrices may also be incorporated into the formulation, e.g., alginates,polysaccharides. Another form of a controlled release of thistherapeutic is by a method based on the Oros therapeutic system (AlzaCorp.), i.e., the drug is enclosed in a semipermeable membrane whichallows water to enter and push drug out through a single small openingdue to osmotic effects. Some enteric coatings also have a delayedrelease effect.

Other coatings may be used for the formulation. These include a varietyof sugars which could be applied in a coating pan. The therapeutic agentcould also be given in a film coated tablet and the materials used inthis instance are divided into 2 groups. The first are the nonentericmaterials and include methyl cellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating.Film coating may be carried out in a pan coater or in a fluidized bed orby compression coating.

Also contemplated herein is pulmonary delivery of the present protein(or derivatives thereof). The protein (or derivative) is delivered tothe lungs of a mammal while inhaling and traverses across the lungepithelial lining to the blood stream. (Other reports of this includeAdjei et al., Pharmaceutical Research 7:565-569 (1990); Adjei et al.,International Journal of Pharmaceutics 63:135-144 (1990)(leuprolideacetate); Braquet et al., Journal of Cardiovascular Pharmacology 13(suppl.5): s.143-146 (1989)(endothelin-1); Hubbard et al., Annals ofInternal Medicine 3:206-212 (1989)(α1-antitrypsin); Smith et al., J.Clin. Invest. 84:1145-1146 (1989)(α1-proteinase); Oswein et al.,“Aerosolization of Proteins”, Proceedings of Symposium on RespiratoryDrug Delivery II, Keystone, Colorado, March, 1990 (recombinant humangrowth hormone); Debs et al., The Journal of Immunology 140:3482-3488(1988)(interferon-γ and tumor necrosis factor α) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor).

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of the inventive compound. Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to diluents, adjuvantsand/or carriers useful in therapy.

The inventive compound should most advantageously be prepared inparticulate form with an average particle size of less than 10 nm (ormicrons), most preferably 0.5 to 5 μm, for most effective delivery tothe distal lung.

Carriers include carbohydrates such as trehalose, mannitol, xylitol,sucrose, lactose, and sorbitol. Other ingredients for use informulations may include DPPC, DOPE, DSPC and DOPC. Natural or syntheticsurfactants may be used. Polyethylene glycol may be used (even apartfrom its use in derivatizing the protein or analog). Dextrans, such ascyclodextran, may be used. Bile salts and other related enhancers may beused. Cellulose and cellulose derivatives may be used. Amino acids maybe used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the inventive compound dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the inventive compoundsuspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the inventive compound and may alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in amounts which facilitate dispersal of thepowder from the device, e.g., 50 to 90% by weight of the formulation.

Nasal delivery of the inventive compound is also contemplated. Nasaldelivery allows the passage of the protein to the blood stream directlyafter administering the therapeutic product to the nose, without thenecessity for deposition of the product in the lung. Formulations fornasal delivery include those with dextran or cyclodextran. Delivery viatransport across other mucous membranes is also contemplated.

Dosages

The dosage regimen involved in a method for treating the above-describedconditions will be determined by the attending physician, consideringvarious factors which modify the action of drugs, e.g. the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. Generally,the dose should be in the range of 0.1 μg to 100 mg of the inventivecompound per kilogram of body weight per day, preferably 0.1 to 1000μg/kg; and more preferably 0.1 to 150 μg/kg, given in daily doses or inequivalent doses at longer or shorter intervals, e.g., every other day,twice weekly, weekly, or twice or three times daily.

The inventive compound may be administered by an initial bolus followedby a continuous infusion to maintain therapeutic circulating levels ofdrug product. As another example, the inventive compound may beadministered as a one-time dose. Those of ordinary skill in the art willreadily optimize effective dosages and administration regimens asdetermined by good medical practice and the clinical condition of theindividual patient. The frequency of dosing will depend on thepharmacokinetic parameters of the agents and the route ofadministration. The optimal pharmaceutical formulation will bedetermined by one skilled in the art depending upon the route ofadministration and desired dosage. See for example, Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa. 18042) pages 1435-1712, the disclosure of which is herebyincorporated by reference. Such formulations may influence the physicalstate, stability, rate of in vivo release, and rate of in vivo clearanceof the administered agents. Depending on the route of administration, asuitable dose may be calculated according to body weight, body surfacearea or organ size. Further refinement of the calculations necessary todetermine the appropriate dosage for treatment involving each of theabove mentioned formulations is routinely made by those of ordinaryskill in the art without undue experimentation, especially in light ofthe dosage information and assays disclosed herein, as well as thepharmacokinetic data observed in the human clinical trials discussedabove. Appropriate dosages may be ascertained through use of establishedassays for determining blood levels dosages in conjunction withappropriate dose-response data. The final dosage regimen will bedetermined by the attending physician, considering various factors whichmodify the action of drugs, e.g. the drug's specific activity, theseverity of the damage and the responsiveness of the patient, the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. As studiesare conducted, further information will emerge regarding the appropriatedosage levels and duration of treatment for various diseases andconditions.

The therapeutic methods, compositions and compounds of the presentinvention may also be employed, alone or in combination with othercytokines, soluble Mpl receptor, hematopoietic factors, interleukins,growth factors or antibodies in the treatment of disease statescharacterized by other symptoms as well as platelet deficiencies. It isanticipated that the inventive compound will prove useful in treatingsome forms of thrombocytopenia in combination with general stimulatorsof hematopoiesis, such as IL-3 or GM-CSF. Other megakaryocyticstimulatory factors, i.e., meg-CSF, stem cell factor (SCF), leukemiainhibitory factor (LIF), oncostatin M (OSM), or other molecules withmegakaryocyte stimulating activity may also be employed with Mpl ligand.Additional exemplary cytokines or hematopoietic factors for such co-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5,IL-6, IL-11, colony stimulating factor-1 (CSF-1), M-CSF, SCF, GM-CSF,granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha(IFN-alpha), consensus interferon, IFN-beta, IFN-gamma, IL-7, IL-8,IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,thrombopoietin (TPO), angiopoietins, for example

Ang-1, Ang-2, Ang-4, Ang-Y, the human angiopoietin-like polypeptide,vascular endothelial growth factor (VEGF), angiogenin, bone morphogenicprotein-1, bone morphogenic protein-2, bone morphogenic protein-3, bonemorphogenic protein- 4, bone morphogenic protein-5, bone morphogenicprotein-6, bone morphogenic protein-7, bone morphogenic protein-8, bonemorphogenic protein-9, bone morphogenic protein-10, bone morphogenicprotein-11, bone morphogenic protein-12, bone morphogenic protein-13,bone morphogenic protein-14, bone morphogenic protein-15, bonemorphogenic protein receptor IA, bone morphogenic protein receptor IB,brain derived neurotrophic factor, ciliary neutrophic factor, ciliaryneutrophic factor receptor a, cytokine-induced neutrophil chemotacticfactor 1, cytokine-induced neutrophil, chemotactic factor 2α,cytokine-induced neutrophil chemotactic factor 2β, β endothelial cellgrowth factor, endothelin 1, epidermal growth factor, epithelial-derivedneutrophil attractant, fibroblast growth factor 4, fibroblast growthfactor 5, fibroblast growth factor 6, fibroblast growth factor 7,fibroblast growth factor 8, fibroblast growth factor 8b, fibroblastgrowth factor 8c, fibroblast growth factor 9, fibroblast growth factor10, fibroblast growth factor acidic, fibroblast growth factor basic,glial cell line-derived neutrophic factor receptor α 1, glial cellline-derived neutrophic factor receptor α 2, growth related protein,growth related protein α, growth related protein β, growth relatedprotein γ, heparin binding epidermal growth factor, hepatocyte growthfactor, hepatocyte growth factor receptor, insulin-like growth factor I,insulin-like growth factor receptor, insulin-like growth factor II,insulin-like growth factor binding protein, keratinocyte growth factor,leukemia inhibitory factor, leukemia inhibitory factor receptor α, nervegrowth factor nerve growth factor receptor, neurotrophin-3,neurotrophin-4, placenta growth factor, placenta growth factor 2,platelet-derived endothelial cell growth factor, platelet derived growthfactor, platelet derived growth factor A chain, platelet derived growthfactor AA, platelet derived growth factor AB, platelet derived growthfactor B chain, platelet derived growth factor BB, platelet derivedgrowth factor receptor α, platelet derived growth factor receptor β,pre-B cell growth stimulating factor, stem cell factor receptor, TNF,including TNF0, TNF1, TNF2, transforming growth factor α, transforminggrowth factor β, transforming growth factor β1, transforming growthfactor β1.2, transforming growth factor β2, transforming growth factorβ3, transforming growth factor β5, latent transforming growth factor β1,transforming growth factor β binding protein I, transforming growthfactor β binding protein II, transforming growth factor β bindingprotein III, tumor necrosis factor receptor type I, tumor necrosisfactor receptor type II, urokinase-type plasminogen activator receptor,vascular endothelial growth factor, and chimeric proteins andbiologically or immunologically active fragments thereof. It may furtherbe useful to administer, either simultaneously or sequentially, aneffective amount of a soluble mammalian Mpl receptor, which appears tohave an effect of causing megakaryocytes to fragment into platelets oncethe megakaryocytes have reached mature form. Thus, administration of aninventive compound (to enhance the number of mature megakaryocytes)followed by administration of the soluble Mpl receptor (to inactivatethe ligand and allow the mature megakaryocytes to produce platelets) isexpected to be a particularly effective means of stimulating plateletproduction. The dosage recited above would be adjusted to compensate forsuch additional components in the therapeutic composition. Progress ofthe treated patient can be monitored by conventional methods.

In cases where the inventive compounds are added to compositions ofplatelets and/or megakaryocytes and related cells, the amount to beincluded will generally be ascertained experimentally by techniques andassays known in the art. An exemplary range of amounts is 0.1 μg-1 mginventive compound per 10⁶ cells.

It is understood that the application of the teachings of the presentinvention to a specific problem or situation will be within thecapabilities of one having ordinary skill in the art in light of theteachings contained herein. Examples of the products of the presentinvention and representative processes for their isolation, use, andmanufacture appear below.

EXAMPLES

I. The following sets forth exemplary methods for making some of thecompounds of the first group disclosed herein.

A. Materials and Methods

All amino acid derivatives (all of L-configurations) and resins used inpeptide synthesis were purchased from Novabiochem. Peptide synthesisreagents (DCC, HOBt, etc.) were purchased in the solution forms fromApplied Biosystems, Inc. The two PEG derivatives were from ShearwaterPolymers, Inc. All solvents (dichloromethane, N-methylpyrrolidinone,methanol, acetonitrile) were from EM Sciences. Analytical HPLC was runon a Beckman system with a Vydac column (0.46 cm×25 cm, C18 reversedphase, 5 mm), at a flow rate of 1 ml/min and with dual UV detection at220 and 280 nm. Linear gradients were used for all HPLC operations withtwo mobile phases: Buffer A—H₂O (0.1% TFA) and Buffer B—acetonitrile(0.1% TFA). The numbered peptides referred to herein, e.g., 17b, 18, 19,and 20, are numbered in reference to Table 1, and some of them arefurther illustrated in FIGS. 2 and 3.

Peptide synthesis. All peptides were prepared by the well establishedstepwise solid phase synthesis method. Solid-phase synthesis with Fmocchemistry was carried out using an ABI Peptide Synthesizer. Typically,peptide synthesis began with a preloaded Wang resin on a 0.1 mmol scale.Fmoc deprotection was carried out with the standard piperidine protocol.The coupling was effected using DCC/HOBt. Side-chain protecting groupswere: Glu(O-t-Bu), Thr(t-Bu), Arg(Pbf), Gln(Trt), Trp(t-Boc) andCys(Trt). For the first peptide precursor for pegylation, Dde was usedfor side chain protection of the Lys on the linker and Boc-Ile-OH wasused for the last coupling. Dde was removed by using anhydrous hydrazine(2% in NMP, 3×2 min), followed by coupling with bromoacetic anhydridepreformed by the action of DCC. For peptide 18, the cysteine side chainin the linker was protected by a trityl group. The final deprotectionand cleavage of all peptidyl-resins was effected at RT for 4 hr, usingtrifluoroacetic acid (TFA) containing 2.5% H₂O, 5% phenol, 2.5%triisopropylsilane and 2.5% thioanisole. After removal of TFA, thecleaved peptide was precipitated with cold anhydrous ether. Disulfideformation of the cyclic peptide was performed directly on the crudematerial by using 15% DMSO in H₂O (pH 7.5). All crude peptides werepurified by preparative reverse phase HPLC and the structures wereconfirmed by ESI-MS and amino acid analysis.

Alternatively, all peptides described above could also be prepared byusing the t-Boc chemistry. In this case, the starting resins would bethe classic Merrifield or Pam resin, and side chain protecting groupswould be: Glu(OBzl), Thr(Bzl), Arg(Tos), Trp(CHO), Cys(p-MeBzl).Hydrogen fluoride (HF) would be used for the final cleavage of thepeptidyl resins.

All the tandem dimeric peptides described in this study that havelinkers composed of natural amino acids can also be prepared byrecombinant DNA technology.

PEGylation. A novel, convergent strategy for the pegylation of syntheticpeptides was developed which consists of combining, through forming aconjugate linkage in solution, a peptide and a PEG moiety, each bearinga special functionality that is mutually reactive toward the other. Theprecursor peptides can be easily prepared with the conventional solidphase synthesis as described above. As described below, these peptidesare “preactivated” with an appropriate functional group at a specificsite. The precursors are purified and fully characterized prior toreacting with the PEG moiety. Ligation of the peptide with PEG usuallytakes place in aqueous phase and can be easily monitored by reversephase analytical HPLC. The pegylated peptides can be easily purified bypreparative HPLC and characterized by analytical HPLC, amino acidanalysis and laser desorption mass spectrometry.

Preparation of peptide 19. Peptide 17b (12 mg) and MeO-PEG-SH 5000 (30mg, 2 equiv.) were dissolved in 1 ml aqueous buffer (pH 8). The mixturewas incubated at RT for about 30 min and the reaction was checked byanalytical HPLC which showed a >80% completion of the reaction. Thepegylated material was isolated by preparative HPLC.

Preparation of peptide 20. Peptide 18 (14 mg) and MeO-PEG-maleimide (25mg) were dissolved in about 1.5 ml aqueous buffer (pH 8). The mixturewas incubated at RT for about 30 min, at which time ˜70% transformationwas complete as monitored with analytical HPLC by applying an aliquot ofsample to the HPLC column. The pegylated material was purified bypreparative HPLC.

Bioactivity assay. The TPO in vitro bioassay is a mitogenic assayutilizing an IL-3 dependent clone of murine 32D cells that have beentransfected with human mpl receptor. This assay is described in greaterdetail in WO 95/26746. Cells are maintained in MEM medium containing 10%Fetal Clone II and 1 ng/ml mIL-3 Prior to sample addition, cells areprepared by rinsing twice with growth medium lacking mIL-3 An extendedtwelve point TPO standard curve is prepared, ranging from 3333 to 39pg/ml. Four dilutions, estimated to fall within the linear portion ofthe standard curve, (1000 to 125 pg/ml), are prepared for each sampleand run in triplicate. A volume of 100 μl of each dilution of sample orstandard is added to appropriate wells of a 96 well microtiter platecontaining 10,000 cells/well. After forty-four hours at 37° C. and 10%CO₂, MTS (a tetrazolium compound which is bioreduced by cells to aformazan) is added to each well. Approximately six hours later, theoptical density is read on a plate reader at 490 nm. A dose responsecurve (log TPO concentration vs. O.D.—Background) is generated andlinear regression analysis of points which fall in the linear portion ofthe standard curve is performed. Concentrations of unknown test samplesare determined using the resulting linear equation and a correction forthe dilution factor.

Abbreviations. HPLC: high performance liquid chromatography; ESI-MS:Electron spray ionization mass spectrometry; MALDI-MS: Matrix-assistedlaser desorption ionization mass spectrometry; PEG: Poly(ethyleneglycol). All amino acids are represented in the standard three-letter orsingle-letter codes. t-Boc: tert-Butoxycarbonyl; tBu: tert-Butyl; Bzl:Benzyl; DCC: Dicylcohexylcarbodiimide; HOBt: 1-Hydroxybenzotriazole;NMP: N-methyl-2-pyrrolidinone; Pbf:2,2,4,6,7-pendamethyldihydro-benzofuran-5-sulfonyl; Trt: trityl; Dde:1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl.

B. Results

TMP tandem dimers with polyglycine linkers. The design of sequentiallylinked TMP dimers was based on the assumption that a dimeric form of TMPwas required for its effective interaction with c-Mpl (the TPO receptor)and that depending on how they were wound up against each other in thereceptor context, the two TMP molecules could be tethered together inthe C- to N-terminus configuration in a way that would not perturb theglobal dimeric conformation.

Clearly, the activity of the tandem linked dimers may also depend onproper selection of the length and composition of the linker that joinsthe C- and -termini of the two sequentially aligned TMP monomers. Sinceno structural information of the TMP bound to c-Mpl was available, aseries of dimeric peptides with linkers composed of 0 to 10 and 14glycine residues (Table 1) were synthesized. Glycine was chosen becauseof its simplicity and flexibility. It was reasoned that a flexiblepolyglycine peptide chain might allow for the free folding of the twotethered TMP repeats into the required conformation, while moresterically hindered amino acid sequences may adopt undesired secondarystructures whose rigidity might disrupt the correct packing of thedimeric peptide in the receptor context.

The resulting peptides are readily accessible by conventional solidphase peptide synthesis methods (Merrifiled, R. B., Journal of theAmerican Chemical Society 85:2149 (1963)) with either Fmoc or t-Bocchemistry. Unlike the synthesis of the C-terminally linked paralleldimer (SEQ ID NO: 2) which required the use of an orthogonally protectedlysine residue as the initial branch point to build the two peptidechains in a pseudosymmetrical way (Cwirla, S. E. et al., Science276:1696-1699 (1997)), the synthesis of our tandem dimers was astraightforward, stepwise assembly of the continuous peptide chains fromthe C- to N-terminus Since dimerization of TMP had a more dramaticeffect on the proliferative activity than binding affinity as shown forthe C-terminal dimer (Cwirla, S. E. et al., Science 276:1696-1699(1997)), the synthetic peptides were tested directly for biologicalactivity in a TPO-dependent cell-proliferation assay using an IL-3dependent clone of murine 32D cells transfected with the full-lengthc-Mpl (Palacios, R. et al., Cell 41:727 (1985)). As the test resultsshowed (see Table 1 below), all of the polyglycine linked tandem dimersdemonstrated >1000 fold increases in potency as compared to the monomer,and were even more potent than the C-terminal dimer in this cellproliferation assay. The absolute activity of the C-terminal dimer inour assay was lower than that of the native TPO protein, which isdifferent from the previously reported findings in which the C-terminaldimer was found to be as active as the natural ligand (Cwirla, S. E. etal., Science 276:1696-1699 (1997)). This might be due to differences inthe conditions used in the two assays. Nevertheless, the difference inactivity between tandem dimers {circle around (c)} terminal of firstmonomer linked to N terminal of second monomer) and parallel dimers{circle around (c)} terminal of first monomer linked to C terminal ofsecond monomer) in the same assay clearly demonstrated the superiorityof tandem dimerized product compared to parallel dimer products. It isinteresting to note that a wide range of length is tolerated by thelinker. The optimal linker with the selected TMP monomers (SEQ ID NO: 1)apparently is composed of 8 glycines.

Other tandem dimers. Subsequent to this first series of TMP tandemdimers, several other molecules were designed either with differentlinkers or containing modifications within the monomer itself The firstof these molecules, peptide 13, has a linker composed of GPNG, asequence known to have a high propensity to form a β-turn-type secondarystructure. Although still about 100-fold more potent than the monomer,this peptide was found to be >10-fold less active than the GGGG-linkedanalog. Thus, introduction of a relatively rigid β-turn at the linkerregion seemed to cause a slight distortion of the optimal agonistconformation in this short linker form.

The Trp9 in the TMP sequence is a highly conserved residue among theactive peptides isolated from random peptide libraries. There is also ahighly conserved Trp in the consensus sequences of EPO mimetic peptidesand this Trp residue was found to be involved in the formation of ahydrophobic core between the two EPO Mimetic Peptides (EMPs) andcontributed to hydrophobic interactions with the EPO receptor (Livnah,O. et al., Science 273:464-471 (1996)). By analogy, it was thought thatthe Trp9 residue in TMP might have a similar function in dimerization ofthe peptide ligand, and in an attempt to modulate and estimate theeffects of noncovalent hydrophobic forces exerted by the two indolerings, several analogs were constructed resulting from mutations at theTrp. So in peptide 14, the Trp residue in each of the two TMP monomerswas replaced with a Cys, and an intramolecular disulfide bond was formedbetween the two cysteines by oxidation which was envisioned to mimic thehydrophobic interactions between the two Trp residues in peptidedimerization. Peptide 15 is the reduced form of peptide 14. In peptide16, the two Trp residues were replaced by Ala. As the assay data show,all three analogs were inactive. These data further demonstrated thatTrp is important for the activity of the TPO mimetic peptide, not justfor dimer formation.

The next two peptides (peptide 17a, and 18) each contain in their8-amino acid linker a Lys or Cys residue. These two compounds areprecursors to the two pegylated peptides (peptide 19 and 20) in whichthe side chain of the Lys or Cys is modified by a polyethylene glycol(PEG) moiety. It was decided to introduce a PEG moiety in the middle ofa relatively long linker, so that the large PEG component (5 kDa) is farenough away from the important binding sites in the peptide molecule.PEG is a known biocompatible polymer which is increasingly used as acovalent modifier to improve the pharmacokinetic profiles of peptide-and protein-based therapeutics.

A modular, solution based method was devised for convenient pegylationof synthetic or recombinant peptides. The method is based on the nowwell established chemoselective ligation strategy which utilizes thespecific reaction between a pair of mutually reactive functionalities.So, for pegylated peptide 19, the lysine side chain was preactivatedwith a bromoacetyl group to give peptide 17b to accommodate reactionwith a thiol-derivatized PEG. To do that, an orthogonal protectinggroup, Dde, was employed for the protection of the lysine ε-amine. Oncethe whole peptide chain was assembled, the N-terminal amine wasreprotected with t-Boc. Dde was then removed to allow for thebromoacetylation. This strategy gave a high quality crude peptide whichwas easily purified using conventional reverse phase HPLC. Ligation ofthe peptide with the thiol-modified PEG took place in aqueous buffer atpH 8 and the reaction completed within 30 min. MALDI-MS analysis of thepurified, pegylated material revealed a characteristic, bell-shapedspectrum with an increment of 44 Da between the adjacent peaks. ForPEG-peptide 20, a cysteine residue was placed in the linker region andits side chain thiol group would serve as an attachment site for amaleimide-containing PEG. Similar conditions were used for thepegylation of this peptide. As the assay data revealed, these twopegylated peptides had even higher in vitro bioactivity as compared totheir unpegylated counterparts.

Peptide 21 has in its 8-amino acid linker a potential glycosylationmotif, NGS. Since the exemplary tandem dimers are made up of naturalamino acids linked by peptide bonds, expression of such a molecule in anappropriate eukaryotic cell system should produce a glycopeptide withthe carbohydrate moiety added on the side chain carboxyamide of Asn.Glycosylation is a common post-translational modification process whichcan have many positive impacts on the biological activity of a givenprotein by increasing its aqueous solubility and in vivo stability. Asthe assay data show, incorporation of this glycosylation motif into thelinker maintained high bioactivity. The synthetic precursor of thepotential glycopeptide had in effect an activity comparable to that ofthe -(Gly)₈-linked analog. Once glycosylated, this peptide is expectedto have the same order of activity as the pegylated peptides, because ofthe similar chemophysical properties exhibited by a PEG and acarbohydrate moiety.

The last peptide is a dimer of a dimer. It was prepared by oxidizingpeptide 18, which formed an intermolecular disulfide bond between thetwo cysteine residues located at the linker. This peptide was designedto address the possibility that TMP was active as a tetramer. The assaydata showed that this peptide was not more active than an average tandemdimer on an adjusted molar basis, which indirectly supports the ideathat the active form of TMP is indeed a dimer, otherwise dimerization ofa tandem dimer would have a further impact on the bioactivity.

The following Table I summarizes relative activities of the above-described compounds in terms of the EC50 based on in vitro assays asdescribed above.

TABLE I Rela- tive Po- Compound tency TPO 4.0 TMP monomer (SEQ ID NO: 1)1.0 TMP C-C dimer (SEQ ID NO: 2) 3.5 TMP-(Gly)_(n)-TMP:  1 n = 0 4.5  2n = 1 4.0  3 n = 2 4.0  4 n = 3 4.0  5 n = 4 4.0  6 n = 5 4.0  7 n = 64.0  8 n = 7 4.0  9 n = 8 4.5 10 n = 9 4.0 11 n = 10 4.0 12 n = 14 4.013 TMP-GPNG-TMP (SEQ ID NO. 10) 3.0 14

0.5 (SEQ ID NO. 11) 15 IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA 0.5 (SEQID NO. 12) 16 IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA 0.5 (SEQ ID NO. 13)17a TMP-GGGKGGGG-TMP (SEQ ID NO. 14) 4.0 17b TMP-GGGK(BrAc)GGGG-TMP (SEQID NO. 15) ND 18 TMP-GGGCGGGG-TMP (SEQ ID NO. 16) 4.0 19TMP-GGGK(PEG)GGGG-TMP (SEQ ID NO. 17) 5.0 20 TMP-GGGC(PEG)GGGG-TMP (SEQID NO. 18) 5.0 21 TMP-GGGNGSGG-TMP (SEQ ID NO. 19) 4.0 22

4.0 NOTE: In Table 1, numerals indicate approximately 1 log of activity,so that the difference in activity between “1” and “4” is approximately1000-fold. An increment of 0.5 is an intermediate point, so that thedifference in activity between “1” and “3.5” is approximately 500-fold.“ND” means not determined

II. The following sets forth exemplary methods for making some of thecompounds of the second group disclosed herein.

A. Preparation of an Fc Fusion Compound of the Type Shown in FIG. 6C.

A DNA sequence coding for the Fc region of human IgG1 fused in-frame toa dimer of the TPO-mimetic peptide (SEQ ID NO: 34) was placed undercontrol of the luxPR promoter in the plasmid expression vector pAMG21 asfollows.

The fusion gene was constructed using standard PCR technology. Templatesfor PCR reactions were the fusion vector containing the Fc sequence anda synthetic gene encoding the remainder of the compound of SEQ ID NO:34. The synthetic gene was constructed from the 4 overlappingoligonucleotides shown below:

1830-52 (SEQ ID NO: 35) AAA GGT GGA GGT GGT GGT ATC GAA GGT CCGACT CTG CGT CAG TGG CTG GCT GCT CGT GCT 1830-53 (SEQ ID NO: 36)ACC TCC ACC ACC AGC ACG AGC AGC CAG CCA CTG ACG CAG AGT CGG ACC 1830-54(SEQ ID NO: 37) GGT GGT GGA GGT GGC GGC GGA GGT ATT GAG GGCCCA ACC CTT CGC CAA TGG CTT GCA GCA CGC GCA 1830-55 (SEQ ID NO: 38)AAA AAA AGG ATC CTC GAG ATT ATG CGC GTG CTGCAA GCC ATT GGC GAA GGG TTG GGC CCT CAA TAC CTC CGC CGC C

The 4 oligonucleotides were annealed to form the duplex shown below:

AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT1--------+---------+---------+---------+---------+---------+ 60                        CCAGGCTGAGACGCAGTCACCGACCGACGAGCACGA K  G  G  G  G  G  I  E  G  P  T  L  R  Q  W  L  A  A  R  AGGTGGTGGAGGTGGCGGCGGAGGTATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCA---------+---------+---------+---------+---------+---------+ 120CCACCACCTCCACCGCCGCCTCCATAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGT G  G  G  G  G  G  G  G  I  E  G  P  L  R  Q  W  L  A  A  A CGCGCA---------------------------148 GCGCGTATTAGAGCTCCTAGGAAAAAAA  R  A   *

SEQ ID NO: 39 [co-linear oligonucleotides 1830-52 and 1830-54]

SEQ ID NO: 40 [co-linear oligonucleotides 1830-53 and 1830-55]

and SEQ ID NO: 41 [the encoded amino acid sequence]

This duplex was amplified in a PCR reaction using 1830-52 and 1830-55 asthe sense and antisense primers.

The Fc portion of the molecule was generated in a PCR reaction with FcDNA using the primers

1216-52 (SEQ ID NO: 42) AAC ATA AGT ACC TGT AGG ATC G 1830-51(SEQ ID NO: 43) TTCGATACCACCACCTCCACCTTTACCCGGAG- ACAGGGAGAGGCTCTTCTGC

The oligonucleotides 1830-51 and 1830-52 contain an overlap of 24nucleotides, allowing the two genes to be fused together in the correctreading frame by combining the above PCR products in a third reactionusing the outside primers, 1216-52 and 1830-55

The final PCR gene product (the full length fusion gene) was digestedwith restriction endonucleases XbaI and BamHI, and then ligated into thevector pAMG21 (see below), also digested with XbaI and BamHI. LigatedDNA was transformed into competent host cells of E. coli strain 2596(GM221, described below). Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. Protein expression levels weredetermined from 50 ml shaker flask studies. Whole cell lysates wereanalyzed for expression of the fusion via Coomassie stained PAGE gels.

The amino acid sequence of the fusion protein is shown below thecorresponding nucleotide sequence:

-   SEQ ID NO: 44 [single strand reading 5′→3′ above],-   SEQ ID NO: 45 [single strand reading 3′→5′ above] and-   SEQ ID NO: 46 [the encoded amino acid sequence] pAMG21

The expression plasmid pAMG21 is available from the ATCC under accessionnumber 98113, which was deposited on Jul. 24, 1996.

GM221 (Amgen Host Strain #2596)

The Amgen host strain #2596 is an E.coli K-12 strain that has beenmodified to contain both the temperature sensitive lambda repressorcI857s7 in the early ebg region and the lacI^(Q) repressor in the lateebg region (68 minutes). The presence of these two repressor genesallows the use of this host with a variety of expression systems,however both of these repressors are irrelevant to the expression fromluxP_(R). The untransformed host has no antibiotic resistances.

The ribosome binding site of the cI857s7 gene has been modified toinclude an enhanced RBS. It has been inserted into the ebg operonbetween nucleotide position 1170 and 1411 as numbered in Genbankaccession number M64441Gb_Ba with deletion of the intervening ebgsequence.

The construct was delivered to the chromosome using a recombinant phagecalled MMebg-cI857s7 enhanced RBS #4 into F′tet/393. After recombinationand resolution only the chromosomal insert described above remains inthe cell. It was renamed F′tet/GM101.

F′tet/GM101 was then modified by the delivery of a lacI^(Q) constructinto the ebg operon between nucleotide position 2493 and 2937 asnumbered in the Genbank accession number M64441Gb_Ba with the deletionof the intervening ebg sequence.

The construct was delivered to the chromosome using a recombinant phagecalled AGebg-LacIQ#5 into F′tet/GM101. After recombination andresolution only the chromosomal insert described above remains in thecell. It was renamed F′tet/GM221. The F′tet episome was cured from thestrain using acridine orange at a concentration of 25 ug/ml in LB. Thecured strain was identified as tetracyline sensitive and was stored asGM221.

The Fc fusion construct contained in plasmid pAMG21 (referred to hereinas pAMG21-Fc-TMP-TMP), which in turn is contained in the host strainGM221 has been deposited at the ATCC under accession number 98957, witha deposit date of Oct. 22, 1998.

Expression. Cultures of pAMG21-Fc-TMP-TMP in E. coli GM221 in LuriaBroth medium containing 50 μg/ml kanamycin were incubated at 37 prior toinduction. Induction of Fc-TMP-TMP gene product expression from theluxPR promoter was achieved following the addition of the syntheticautoinducer N-(3-oxohexanoyl)-DL-homoserine lactone to the culture mediato a final concentration of 20 ng/ml and cultures were incubated at 37°C. for a further 3 hours. After 3 hours, the bacterial cultures wereexamined by microscopy for the presence of inclusion bodies and werethen collected by centrifugation. Refractile inclusion bodies wereobserved in induced cultures indicating that the Fc-TMP-TMP was mostlikely produced in the insoluble fraction in E. coli. Cell pellets werelysed directly by resuspension in Laemmli sample buffer containing 10%β-mercaptoethanol and were analyzed by SDS-PAGE. An intense Coomassiestained band of approximately 30 kDa was observed on an SDS-PAGE gel.The expected gene product would be 269 amino acids in length and have anexpected molecular weight of about 29.5 kDa. Fermentation was alsocarried out under standard batch conditions at the 10 L scale, resultingin similar expression levels of the Fc-TMP-TMP to those obtained atbench scale.

Purification of Fc-TMP-TMP.

Cells were broken in water (1/10) by high pressure homogenization (2passes at 14,000 PSI) and inclusion bodies were harvested bycentrifugation (4200 RPM in J-6B for 1 hour). Inclusion bodies weresolubilized in 6 M guanidine, 50 mM Tris, 8 mM DTT, pH 8.7 for 1 hour ata 1/10 ratio. The solubilized mixture was diluted 20 times into 2 Murea, 50 mM Tris, 160 mM arginine, 3 mM cysteine, pH 8.5. The mixturewas stirred overnight in the cold. At this point in the procedure theFc-TMP-TMP monomer subunits dimerize to form the disulfide-linkedcompound having the structure shown in FIG. 6C. and then concentratedabout 10 fold by ultafiltration. It was then diluted 3 fold with 10 mMTris, 1.5 M urea, pH 9. The pH of this mixture was then adjusted to pH 5with acetic acid. The precipitate was removed by centrifugation and thesupernatant was loaded onto a SP-Sepharose Fast Flow column equilibratedin 20 mM NaAc, 100 mM NaCl, pH 5(10 mg/ml protein load, roomtemperature). The protein was eluted off using a 20 column volumegradient in the same buffer ranging from 100 mM NaCl to 500 mM NaCl. Thepool from the column was diluted 3 fold and loaded onto a SP-SepharoseHP column in 20 mM NaAc, 150 mM NaCl, pH 5 (10 mg/ml protein load, roomtemperature). The protein was eluted off using a 20 column volumegradient in the same buffer ranging from 150 mM NaCl to 400 mM NaCl. Thepeak was pooled and filtered.

III. The following is a summary of in vivo data in mice with variouscompounds of this invention.

Mice. Normal female BDF1 approximately 10-12 weeks of age.

Bleed schedule. Ten mice per group treated on day 0, two groups started4 days apart for a total of 20 mice per group. Five mice bled at eachtime point, mice were bled a minimum of three times a week. Mice wereanesthetized with isoflurane and a total volume of 140-160 μl of bloodwas obtained by puncture of the orbital sinus. Blood was counted on aTechnicon HIE blood analyzer running software for murine blood.Parameters measured were white blood cells, red blood cells, hematocrit,hemoglobin, platelets, neutrophils.

Treatments. Mice were either injected subcutaneously for a bolustreatment or implanted with 7 day micro-osmotic pumps for continuousdelivery. Subcutaneous injections were delivered in a volume of 0.2 ml.Osmotic pumps were inserted into a subcutaneous incision made in theskin between the scapulae of anesthetized mice. Compounds were dilutedin PBS with 0.1% BSA. All experiments included one control group,labeled “carrier” that were treated with this diluent only. Theconcentration of the test articles in the pumps was adjusted so that thecalibrated flow rate from the pumps gave the treatment levels indicatedin the graphs.

Compounds. A dose titration of the compound was delivered to mice in 7day micro-osmotic pumps. Mice were treated with various compounds at asingle dose of 100 ug/kg in 7 day osmotic pumps. Some of the samecompounds were then given to mice as a single bolus injection.

Activity test results. The results of the activity experiments are shownin FIGS. 4 and 5. In dose response assays using 7-day micro-osmoticpumps (data not shown) the maximum effect was seen with the compound ofSEQ ID NO: 18 was at 100 μg/kg/day; the 10 μg/kg/day dose was about 50%maximally active and 1 μg/kg/day was the lowest dose at which activitycould be seen in this assay system. The compound at 10 μg/kg/day dosewas about equally active as 100 μg/kg/day unpegylated rHu-MGDF in thesame experiment.

IV. Discussion

It is well accepted that MGDF acts in a way similar to human growthhormone (hGH), i.e., one molecule of the protein ligand binds twomolecules of the receptor for its activation (Wells, J. A. et al., Ann.Rev. Biochem. 65:609-634 (1996))). This interaction is mimicked by theaction of the much smaller TMP peptide. However, the present studiessuggest that this mimicry requires the concerted action of two TMPmolecules, as covalent dimerization of TMP in either a C—C parallel orC—N sequential fashion increased the in vitro biological potency of theoriginal monomer by a factor of greater than 10³. The relatively lowbiopotency of the monomer is probably due to inefficient formation ofthe noncovalent dimer A preformed covalent dimer has the ability toeliminate the entropy barrier for the formation of a noncovalent dimerwhich is exclusively driven by weak, noncovalent interactions betweentwo molecules of the small, 14- residue peptide.

It is interesting to note that most of the tandem dimers are more potentthan the C-terminal parallel dimers. Tandem dimerization seems to givethe molecule a better fit conformation than does the C—C paralleldimerization. The seemingly unsymmetric feature of a tandem dimer mighthave brought it closer to the natural ligand which, as an unsymmetricmolecule, uses two different sites to bind two identical receptormolecules.

Introduction of the PEG moiety was envisaged to enhance the in vivoactivity of the modified peptide by providing it a protection againstproteolytic degradation and by slowing down its clearance through renalfiltration. It was unexpected that pegylation could further increase thein vitro bioactivity of a tandem dimerized TMP peptide in the cell-basedproliferation assay.

V. The following is a summary of in vivo data in monkeys with variouscompounds of this invention.

In order to evaluate hematological parameters in female rhesus monkeysassociated with administration of AMP2 via subcutaneous administration,the following protocol was designed and carried out. Five groups ofthree monkeys each were assembled. Group 1 served as control andreceived acetate buffer (20 mM sodium acetate, 0.25 M sodium chloride,pH 5) containing neither AMP2 nor pegylated, recombinant human MGDF(PEG-rHuMGDF). Group 2 received one or more dosage of AMP2 at intervalsindicated below; Group 3 received 1000 μg/kg AMP2 at intervals indicatedbelow; Group 4 received 5000 μg/kg AMP2 at intervals indicated below;and Group 5 received 100 μg/kg PEG-rHuMGDF at intervals indicated below.

The day on which the first single dose was administered was designatedas Day 0 of Cycle 1. In Cycle 2, doses were administered on Days 21, 23,25, 28, 30 and 32. During Cycle 3, a single dose was administered on Day84, and in Cycle 4, a single dose was administration on Day 123. Animalswere observed for clinical signs once daily during the acclimationperiod, three times daily (prior to dosing, immediately to 30 minutesfollowing dosing, and 2 to 3 hours following dosing) on the dosing days,and once daily on the non-dosing days. Food consumption was calculateddaily based on the number of food pieces given and the number left overfor each animal from 7 days prior to the initiation of the dosing periodto the end of the recovery period. Body weight for each animal wasmeasured twice prior to the dosing regimen and twice during the dosingand recovery periods. Blood samples for hematology were prepared onceprior to the initiation of dosing and once on Days 1, 3, 5, 7, 9, 11,13, 15, 20, 22, 24, 26, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 55,62, 69, 76, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 111, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 150. Forpharmacokinetic analysis. 0.5 ml serum samples were collected once priorto dosing and once at 1, 4 and 24 hours after dosing. Samples werecollected on Days 0, 21, 32, 84, and 123 and stored at approximately−70° C. until analysis. For antibody analysis, 2 ml blood samples werecollected one week prior to the single dose and once on Days 0 (prior todosing), 6, 13, 20, 27, 34, 41, 48, 55, 62, 69, 76, 83, 90, 97, 104,111, 118, 129, 136, 143 and 150. Samples were stored at −70° C. untilanalysis.

Results indicated that platelet values increased in all treated groupswith the largest increases seen in the PEG-rHuMGDF and high dose AMP2groups. In Cycle 1, peak platelet values increased approximately3.3-fold and 3.1-fold in the PEG-rHuMGDF group (Day 9) and 5000 μg/kgAMP2 group ((Day 9), respectively, compared to the mean platelet countin the control group. The low dose AMP2 platelet values increasedapproximately 1.5-fold higher than control on the same specified studydays. Similar responses were noted in all other cycles.

However, in Cycle 4, the PEG-rHuMGDF group did not demonstrate as largeof an increased platelet count as in the previous cycles. ThePEG-rHuMGDF group has increased platelet counts of approximately 2-foldthat in the control group 9 days after the dose of this cycle. Forcomparison, the mean platelet count in the highest dose AMP2 group inCycle 4 was 3.3-fold higher than the control group. Additionally,PEG-rHuMGDF animals has a mean platelet count 53% lower than the controlgroup mean platelet count at the start of Cycle 4 (per dose) and themean platelet count for the group at the end of Cycle 4 *(27 days postdose) was 79% lower than that of the control group. For all AMP2animals, the mean platelet counts at the start and end of Cycle 4 were±15% of the platelet count in the control group.

In Cycle 1 and 2, a trend toward a decrease in red blood cell (RBC)counts was noted in all treated groups as compared to control. Thedecrease was most evident by Days 41 to 43 and the largest decrease inRBC was noted in the PEG-rHuMGDF group. The counts began returning tonormal levels (as compared to control) as early as Day 47. The whiteblood cell (WBC) levels during Cycles 1 and 2 were dramaticallyincreased (2.6-fold) as compared to control on Day 35. A slight increasewas noted in the 5000 μg/kg AMP2 group on Day 33. Values headed towardnormal (control) levels beginning on Day 37. A similar response was seenin Cycle 3 with no apparent change in WBC in Cycle 4 in any of thetreated groups.

During Cycle 3, RBC counts were slightly decreased by Day 13 (followingthe single Cycle 3 dose) in all treated groups except for the 500 μg/kgAMP2 group. RBC values began returning to normal levels (as compared tocontrol) by Day 17.

In Cycle 4, RBC counts decreased in all treated groups as compared tocontrol except in the 500 μg/kg AMP2 group. Unlike the other cycles,there was more than one nadir present in this cycle. These decreasesappeared from Day 1-9 post dose and began to recover as early as Day 11.

The results indicated that an increase in platelet counts, above that ofcontrol animals could be detected 7 to 9 days following dosing in alltreated animals in all cycles tested. It appeared that the repeated dosephase caused a higher response in platelet production as compared to thesingle dose phases. By Cycle 4, the platelet response elicited by thePEG-rHuMGDF group was lower compared to the previous cycles and comparedto that of the high dose AMP2 response. Decreases in RBC counts werenoted in Cycles 1, 2, 3 and 4 in most treated groups at some pointduring each cycle of the study, however, all hematology parametersreturned to normal levels (as compared to control) after dosingcessation.

Overall, these results indicated that treatment with AMP2 was welltolerated in the rhesus monkeys and that AMP2 resulted in increasedplatelet counts after various cycles of treatment. It did not appear,based on the platelet count results, that there was a biologicallysignificant immune-mediated response to AMP2. In contrast, treatment inthe various cycles with PEG-rHuMGDF did show an inhibition in plateletresponse by Cycle 4, suggesting that antibodies to PEG-rHuMGDF have beengenerated and these anti-MGDF antibodies may be crossreacting withendogenous rhesus TPO.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto, without departing from the spirit and scope of theinvention as set forth herein.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of producing acompound comprising growing a host cell comprising a polynucleotide thatencodes said compound in a suitable nutrient medium and isolating saidcompound from said cell or nutrient medium, wherein said compound bindsto an mpl receptor and comprises the structureTMP₁-(L₁)_(n)-TMP₂ wherein the C-terminus of the TMP₁ peptide is linkedto the N-terminus of the TMP₂ peptide, optionally via L₁, wherein TMP₁and TMP₂ are each independently selected from the group of corecompounds comprising the structure selected from the group consistingof: a) X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀, wherein, X₂ is selected from thegroup consisting of Glu, Lys, and Val; X₃ is selected from the groupconsisting of Gly and Ala; X₄ is Pro; X₅ is selected from the groupconsisting of Thr and Ser; X₆ is selected from the group consisting ofLeu, Ile, Val, Ala, and Phe; X₇ is selected from the group consisting ofArg and Lys; X₈ is selected from the group consisting of Gln, Asn, andGlu; X₉ is selected from the group consisting of Trp, Tyr, Cys, Ala, andPhe; X₁₀ is selected from the group consisting of Leu, Ile, Val, Ala,Phe, Met, and Lys; b) X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀, wherein, X₂ isselected from the group consisting of Glu, Asp, Lys, and Val; X₃ is Ala;X₄ is Pro; X₅ is selected from the group consisting of Thr and Ser; X₆is selected from the group consisting of Leu, Ile, Val, Ala, and Phe; X₇is selected from the group consisting of Arg and Lys; X₈ is selectedfrom the group consisting of Gln, Asn, and Glu; X₉ is selected from thegroup consisting of Trp, Tyr, Cys, Ala, and Phe; X₁₀ is selected fromthe group consisting of Leu, Ile, Val, Ala, Phe, Met, and Lys; c)X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀, wherein, X₂ is selected from the groupconsisting of Glu, Asp, Lys, and Val; X₃ is selected from the groupconsisting of Gly and Ala; X₄ is Pro; X₅ is Ser; X₆ is selected from thegroup consisting of Leu, Ile, Val, Ala, and Phe; X₇ is selected from thegroup consisting of Arg and Lys; X₈ is selected from the groupconsisting of Gln, Asn, and Glu; X₉ is selected from the groupconsisting of Trp, Tyr, Cys, Ala, and Phe; X₁₀ is selected from thegroup consisting of Leu, Ile, Val, Ala, Phe, Met, and Lys; d)X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀, wherein, X₂ is selected from the groupconsisting of Glu, Asp, Lys, and Val; X₃ is selected from the groupconsisting of Gly and Ala; X₄ is Pro; X₅ is selected from the groupconsisting of Thr and Ser; X₆ is selected from the group consisting ofIle, Val, Ala, and Phe; X₇ is selected from the group consisting of Argand Lys; X₈ is selected from the group consisting of Gln, Asn, and Glu;X₉ is selected from the group consisting of Trp, Tyr, Cys, Ala, and Phe;X₁₀ is selected from the group consisting of Leu, Ile, Val, Ala, Phe,Met, and Lys; e) X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀, wherein, X₂ is selectedfrom the group consisting of Glu, Asp, Lys, and Val; X₃ is selected fromthe group consisting of Gly and Ala; X₄ is Pro; X₅ is selected from thegroup consisting of Thr and Ser; X₆ is selected from the groupconsisting of Leu, Ile, Val, Ala, and Phe; X₇ is Lys; X₈ is selectedfrom the group consisting of Gln, Asn, and Glu; X₉ is selected from thegroup consisting of Trp, Tyr, Cys, Ala, and Phe; X₁₀ is selected fromthe group consisting of Leu, Ile, Val, Ala, Phe, Met, and Lys; f)X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀, wherein, X₂ is selected from the groupconsisting of Glu, Asp, Lys, and Val; X₃ is selected from the groupconsisting of Gly and Ala; X₄ is Pro; X₅ is selected from the groupconsisting of Thr and Ser; X₆ is selected from the group consisting ofLeu, Ile, Val, Ala, and Phe; X₇ is selected from the group consisting ofArg and Lys; X₈ is selected from the group consisting of Gln and Asn; X₉is selected from the group consisting of Trp, Tyr, Cys, Ala, and Phe;X₁₀ is selected from the group consisting of Leu, Ile, Val, Ala, Phe,Met, and Lys; g) X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀, wherein, X₂ is selectedfrom the group consisting of Glu, Asp, Lys, and Val; X₃ is selected fromthe group consisting of Gly and Ala; X₄ is Pro; X₅ is selected from thegroup consisting of Thr and Ser; X₆ is selected from the groupconsisting of Leu, Ile, Val, Ala, and Phe; X₇ is selected from the groupconsisting of Arg and Lys; X₈ is selected from the group consisting ofGln, Asn, and Glu; X₉ is selected from the group consisting of Tyr, Cys,Ala, and Phe; X₁₀ is selected from the group consisting of Leu, Ile,Val, Ala, Phe, Met, and Lys; and h) X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀,wherein, X₂ is selected from the group consisting of Glu, Asp, Lys, andVal; X₃ is selected from the group consisting of Gly and Ala; X₄ is Pro;X₅ is selected from the group consisting of Thr and Ser; X₆ is selectedfrom the group consisting of Leu, Ile, Val, Ala, and Phe; X₇ is selectedfrom the group consisting of Arg and Lys; X₈ is selected from the groupconsisting of Gln, Asn, and Glu; X₉ is selected from the groupconsisting of Trp, Tyr, Cys, Ala, and Phe; X₁₀ is selected from thegroup consisting of Leu, Val, Ala, Phe, Met, and Lys; and wherein L₁ isa linker; and n is 0 or 1, wherein when n is 1, L₁ is independentlyselected from the linker groups consisting of Y_(n), wherein Y is anaturally occurring amino acid or a stereoisomer thereof and n is 1through 20; and physiologically acceptable salts thereof.
 2. The methodaccording to claim 1 wherein said TMP₁ and TMP₂ are independentlyselected from the group consisting of: X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀₋X₁₁;X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀₋X₁₁₋X₁₂;X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀₋X₁₁₋X₁₂₋X₁₃;X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀₋X₁₁₋X₁₂₋X₁₃₋X₁₄;X₁₋X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀; X₁₋X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀₋X₁₁;X₁₋X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀₋X₁₁₋X₁₂;X₁₋X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀X₁₁₋X₁₂₋X₁₃; andX₁₋X₂₋X₃₋X₄₋X₅₋X₆₋X₇₋X₈₋X₉₋X₁₀₋X₁₁₋X₁₂₋X₁₃₋X₁₄, wherein X₂-X₁₀ are asdefined and X₁ and X₁₁-X₁₄ are selected from the group consisting of: a)X₁ is selected from the group consisting of Ile, Val, Leu, Ser, and Arg;X₁₁ is selected from the group consisting of Ala, Ile, Val, Leu, Phe,Ser, Thr, Lys, His, and Glu; X₁₂ is selected from the group consistingof Ala, Ile, Val, Leu, Phe, Gly, Ser, and Gln; X₁₃ is selected from thegroup consisting of Arg, Lys, Thr, Val, Asn, Gln, and Gly; and X₁₄ isselected from the group consisting of Ala, Ile, Val, Leu, Phe, Thr, Arg,Glu, and Gly; b) X₁ is selected from the group consisting of Ile, Ala,Val, Leu, Ser, and Arg; X₁₁ is selected from the group consisting ofAla, Ile, Val, Leu, Phe, Thr, Lys, His, and Glu; X₁₂ is selected fromthe group consisting of Ala, Ile, Val, Leu, Phe, Gly, Ser, and Gln; X₁₃is selected from the group consisting of Arg, Lys, Thr, Val, Asn, Gln,and Gly; and X₁₄ is selected from the group consisting of Ala, Ile, Val,Leu, Phe, Thr, Arg, Glu, and Gly; and c) X₁ is selected from the groupconsisting of Ile, Ala, Val, Leu, Ser, and Arg; X₁₁ is selected from thegroup consisting of Ala, Ile, Val, Leu, Phe, Ser, Thr, Lys, His, andGlu; X₁₂ is selected from the group consisting of Ala, Ile, Val, Leu,Gly, Ser, and Gln; X₁₃ is selected from the group consisting of Arg,Lys, Thr, Val, Asn, Gln, and Gly; and X₁₄ is selected from the groupconsisting of Ala, Ile, Val, Leu, Phe, Thr, Arg, Glu, and Gly.
 3. Themethod according to claim 1 wherein said TMP₁ and/or TMP₂ arederivatized as set forth in one or more of the following: one or more ofthe peptidyl [—C(O)NR—] linkages (bonds) have been replaced by anon-peptidyl linkage selected from the group consisting of: a—CH₂₋carbamate linkage [—CH₂₋OC(O)NR—]; a phosphonate linkage; a—CH₂₋sulfonamide [—CH₂₋S(O)₂NR—] linkage; a urea [—NHC(O)NH—] linkage; a—CH₂₋secondary amine linkage; and an alkylated peptidyl linkage[—C(O)NR⁶⁻ where R⁶ is lower alkyl]; the N-terminus is a —NRR¹ group; toa —NRC(O)R group; to a —NRC(O)OR group; to a —NRS(O)₂R group; to a—NHC(O)NHR group where R and R¹ are hydrogen and lower alkyl with theproviso that R and R¹ are not both hydrogen; to a succinimide group; toa benzyloxycarbonyl-NH—(CBZ—NH—) group; or to a benzyloxycarbonyl-NH—group having from 1 to 3 substituents on the phenyl ring selected fromthe group consisting of lower alkyl, lower alkoxy, chloro, and bromo;the C terminus is —C(O)R² where R² is selected from the group consistingof lower alkoxy and —NR³R⁴ where R³ and R⁴ are independently selectedfrom the group consisting of hydrogen and lower alkyl.
 4. The methodaccording to claim 1 wherein L₁ comprises (Gly)_(n), wherein n is 1through 20, and when n is greater than 1, up to half of the Gly residuesmay be substituted by another amino acid selected from the remaining 19natural amino acids or a stereoisomer thereof.
 5. The method accordingto claim 4 wherein L₁ comprises (Gly)_(n), wherein n is 1 through
 14. 6.The method according to claim 4 wherein L₁ comprises (Gly)₈.
 7. Themethod according to claim 1 wherein L₁ is selected from the groupconsisting of (Gly)₃Lys(Gly)₄ (SEQ ID NO: 6); (Gly)₃AsnGlySer(Gly)₂ (SEQID NO: 7); (Gly)₃Cys(Gly)₄ (SEQ ID NO: 8); and GlyProAsnGly (SEQ ID NO:9).
 8. The method according to claim 1 wherein L₁ comprises a Cysresidue.
 9. The method according to claim 1 wherein L₁ comprises(CH₂)_(n), wherein n is 1 through
 20. 10. The method according to claim1 wherein said polynucleotide is comprised in a vector.
 11. The methodaccording to claim 10 wherein said vector.is plasmid pAMG21 deposited atthe ATCC under accession number
 98113. 12. The method according to claim1 wherein said host cell is selected from bacteria, yeast, fungi, insector mammalian cells.
 13. The method according to claim 12 wherein saidhost cell is E. coli.
 14. The method according to claim 13 wherein saidE. coli. is strain GM221 deposited at the ATCC under accession number98957.